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Aims: Proper arteriogenesis after tissue ischaemia is necessary to rebuild stable blood circulation; nevertheless, this process is impaired in type 2 diabetes mellitus (T2DM). Raptor is a scaffold protein and a component of mammalian target of rapamycin complex 1 (mTORC1). However, the role of the endothelial Raptor in arteriogenesis under the conditions of T2DM remains unknown. This study investigated the role of endothelial Raptor in ischaemia-induced arteriogenesis during T2DM., Methods and Results: Although endothelial mTORC1 is hyperactive in T2DM, we observed a marked reduction in the expression of endothelial Raptor in two mouse models and in human vessels. Inducible endothelial-specific Raptor knockout severely exacerbated impaired hindlimb perfusion and arteriogenesis after hindlimb ischaemic injury in 12-week high-fat diet fed mice. Additionally, we found that Raptor deficiency dampened vascular endothelial growth factor receptor 2 (VEGFR2) signalling in endothelial cells (ECs) and inhibited VEGF-induced cell migration and tube formation in a PTP1B-dependent manner. Furthermore, mass spectrometry analysis indicated that Raptor interacts with neuropilin 1 (NRP1), the co-receptor of VEGFR2, and mediates VEGFR2 trafficking by facilitating the interaction between NRP1 and Synectin. Finally, we found that EC-specific overexpression of the Raptor mutant (loss of mTOR binding) reversed impaired hindlimb perfusion and arteriogenesis induced by endothelial Raptor knockout in high-fat diet fed mice., Conclusion: Collectively, our study demonstrated the crucial role of endothelial Raptor in promoting ischaemia-induced arteriogenesis in T2DM by mediating VEGFR2 signalling. Thus, endothelial Raptor is a novel therapeutic target for promoting arteriogenesis and ameliorating perfusion in T2DM., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Skeletal muscle atrophy and dysfunction commonly accompany cardiovascular diseases such as peripheral arterial disease and may be partially attributable to systemic inflammation. We sought to determine whether acute systemic inflammation in a model of hindlimb ischaemia (HLI) could affect skeletal muscle macrophage infiltration, fibre size, or capillarization, independent of the ischaemia. Eight-week-old C57BL/6 male mice underwent either Sham or HLI surgery, and were killed 1, 3, or 7 days post-surgery. Circulating inflammatory cytokine concentrations were measured, as well as immune cell infiltration and morphology of skeletal muscle from both limbs of HLI and Sham mice. In HLI compared with Sham mice at day 1, plasma interleukin-1β levels were 216% higher (0.48 ± 0.10 vs. 0.15 ± 0.01 pg/μL, P = 0.005) and decreased by day 3. This was followed by increased macrophage presence in muscle from both ischaemic and non-ischaemic limbs of HLI mice by day 7 (7.3- and 2.3-fold greater than Sham, respectively, P < 0.0001). In HLI mice, muscle from the ischaemic limb had 21% lower fibre cross-sectional area than the non-ischaemic limb (724 ± 28 vs. 916 ± 46 μm 2 , P = 0.01), but the non-ischaemic limb of HLI mice was no different from Sham. This shows that HLI induces acute systemic inflammation accompanied by immune infiltration in both ischaemic and remote skeletal muscle; however, this did not induce skeletal muscle atrophy in remote muscle within the 7-day time course of this study. This effect of local skeletal muscle ischaemia on the inflammatory status of remote skeletal muscle may signal a priming of muscle for subsequent atrophy over a longer time course. HIGHLIGHTS: What is the central question of this study? Does hindlimb ischaemia-induced inflammation cause acute immune, inflammatory and morphological alterations in remote non-ischaemic skeletal muscle? What is the main finding and its importance? Hindlimb ischaemia induced systemic inflammation with subsequent neutrophil and macrophage infiltration in both ischaemic and non-ischaemic skeletal muscle; however, morphological changes did not occur in non-ischaemic muscle within 7 days. These immune alterations may have functional implications that take longer than 7 days to manifest, and subsequent or prolonged systemic inflammation and immune infiltration of muscle could lead to morphological changes and functional decline., (© 2024 The Author(s). Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Objectives: The study aimed to determine if an ultrasonic Doppler-guided technique (UDGT) leads to improved placement efficacy (time, success) of feline dorsal pedal arterial catheters vs the traditional palpation-guided technique (TPT)., Methods: A total of 26 adult, client-owned cats requiring sedation or general anesthesia for any reason, aged >12 months and weighing >3.0 kg, and with Doppler blood pressure measurements of at least 80 mmHg were enrolled. Each hindlimb was randomly assigned for dorsal pedal arterial catheterization using either the UDGT or TPT. With the UDGT, the location of the artery was identified by an audible sound using the Doppler. Successful catheter placement was confirmed by visualization of an arterial pressure waveform using a transducer and monitor system attached to the catheter. The Kaplan-Meier method and log-rank test were used to compare the two techniques., Results: The overall proportion of successful arterial catheterization was 17% (9/52): 19% (5/26) via UDGT and 15% (4/26) via TPT. Among successful arterial catheterizations (n = 9), the mean time to catheterization was 339 ± 198 s: 328 ± 237 s (n = 5) with UDGT and 353 ± 171 s (n = 4) with TPT. The log-rank test showed the two techniques were not significantly different in likelihood of successful arterial catheter placement or time to successful catheterization ( P  = 0.698). An arterial flash occurred in 62% (32/52) of the limbs, 58% (15/26) with the UDGT and 65% (17/26) with the TPT. Complications (self-limiting bruising, hematoma formation) were observed equally between UDGT (3/26 limbs) and TPT (3/26 limbs) in six cats., Conclusions and Relevance: The UDGT did not improve the efficacy of catheter placement compared with the TPT. Few complications were associated with arterial catheterization., Competing Interests: Conflict of interestThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Critical limb ischemia (CLI) is a state of severe peripheral artery disease, with no effective treatment. Cell therapy has been investigated as a therapeutic tool for CLI, and pericytes are promising therapeutic candidates based on their angiogenic properties. We firstly generated highly proliferative and immunosuppressive pericyte-like cells from embryonic stem (ES) cells. In order to enhance the angiogenic potential, we transduced the basic fibroblast growth factor (bFGF) gene into the pericyte-like cells and found a significant enhancement of angiogenesis in a Matrigel plug assay. Furthermore, we evaluated the bFGF-expressing pericyte-like cells in the previously established chronic hindlimb ischemia model in which bone marrow-derived MSCs were not effective. As a result, bFGF-expressing pericyte-like cells significantly improved blood flow in both laser Doppler perfusion imaging (LDPI) and dynamic contrast-enhanced MRI (DCE-MRI). These findings suggest that bFGF-expressing pericyte-like cells differentiated from ES cells may be a therapeutic candidate for CLI., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 (ELA ECKO ) 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 ELA ECKO mice and ELA WT mice. After ischemia, the expression of VEGFR2, p-VEGFR2, and p-AKT was significantly lower in ELA ECKO mice than in ELA WT 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A novel NIR-II nanoprobe was developed through regulating the steric effect of an A-DA'D-A dye. The probe features the properties of strong fluorescence, high stability, and a large Stokes shift, thereby serving as a remarkable contrast agent for the fluorescence imaging of hindlimb vasculature and tumors in live mice.

Background: This study explores the potential role of Thioredoxin-interacting protein (TXNIP) silencing in endothelial colony-forming cells (ECFCs) within the scope of age-related comorbidities and impaired vascular repair. We aim to elucidate the effects of TXNIP silencing on vasculogenic properties, paracrine secretion, and neutrophil recruitment under conditions of metabolic stress., Methods: ECFCs, isolated from human blood cord, were transfected with TXNIP siRNA and exposed to a high glucose and β-hydroxybutyrate (BHB) medium to simulate metabolic stress. We evaluated the effects of TXNIP silencing on ECFCs' functional and secretory responses under these conditions. Assessments included analyses of gene and protein expression profiles, vasculogenic properties, cytokine secretion and neutrophil recruitment both in vitro and in vivo. The in vivo effects were examined using a murine model of hindlimb ischemia to observe the physiological relevance of TXNIP modulation under metabolic disorders., Results: TXNIP silencing did not mitigate the adverse effects on cell recruitment, vasculogenic properties, or senescence induced by metabolic stress in ECFCs. However, it significantly reduced IL-8 secretion and consequent neutrophil recruitment under these conditions. In a mouse model of hindlimb ischemia, endothelial deletion of TXNIP reduced MIP-2 secretion and prevented increased neutrophil recruitment induced by age-related comorbidities., Conclusions: Our findings suggest that targeting TXNIP in ECFCs may alleviate ischemic complications exacerbated by metabolic stress, offering potential clinical benefits for patients suffering from age-related comorbidities., (© 2024. The Author(s).)

MicroRNAs (miRs) regulate physiological and pathological processes, including ischemia-induced angiogenesis and neovascularization. They can be transferred between cells by extracellular vesicles (EVs). However, the specific miRs that are packaged in EVs released from skeletal muscles, and how this process is modulated by ischemia, remain to be determined. We used a mouse model of hindlimb ischemia and next generation sequencing (NGS) to perform a complete profiling of miR expression and determine the effect of ischemia in skeletal muscles, and in EVs of different sizes (microvesicles (MVs) and exosomes) released from these muscles. Ischemia significantly modulated miR expression in whole muscles and EVs, increasing the levels of several miRs that can have pro-angiogenic effects (angiomiRs). We found that specific angiomiRs are selectively enriched in MVs and/or exosomes in response to ischemia. In silico approaches indicate that these miRs modulate pathways that play key roles in angiogenesis and neovascularization, including HIF1/VEGF signaling, regulation of actin cytoskeleton and focal adhesion, NOTCH, PI3K/AKT, RAS/MAPK, JAK/STAT, TGFb/SMAD signaling and the NO/cGMP/PKG pathway. Thus, we show for the first time that angiomiRs are selectively enriched in MVs and exosomes released from ischemic muscles. These angiomiRs could be targeted in order to improve the angiogenic function of EVs for potential novel therapeutic applications in patients with severe ischemic vascular diseases.

Intravenous regional limb perfusion (IVRLP) has been used in the treatment of pododermatitis and distal limb infections, which are significant causes of morbidity in avian species. This intravenous drug administration technique is designed to achieve high drug tissue concentrations while minimizing systemic toxic effects. Amikacin is commonly used for IVRLP in veterinary medicine, but dosing guidelines have not been established for its use in birds. The current study aimed to determine the tissue concentration of amikacin after a single IVRLP administration in healthy, euhydrated leghorn hen chickens ( Gallus gallus domesticus ). Chickens received a single IVRLP dose of 10 mg/kg amikacin and were euthanatized posttreatment at 1 hour (n = 6), 12 hours (n = 6), and 24 hours (n = 6) to assess tissue and synovial fluid concentrations of amikacin in the injected leg. Mean tissue concentrations were highest 1 hour post-IVRLP (synovial fluid = 153.0 µg/mL, metatarsal pad tissue = 26.05 µg/mL) before declining at the 12- and 24-hour time points. This indicates that administration of amikacin via IVRLP can reach minimum inhibitory concentrations of common bacterial isolates in tissues after a single treatment with 10 mg/kg amikacin. Regional limb perfusion every 24 hours is recommended, although the minimum days of treatment may be case dependent and vary based on response to therapy.

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.

Sterile inflammation after injury is important for tissue restoration. In injured human and mouse tissues, macrophages were recently found to accumulate perivascularly. This study investigates if macrophages adopt a mural cell phenotype important for restoration after ischemic injury. Single-cell RNA sequencing of fate-mapped macrophages from ischemic mouse muscles demonstrates a macrophage-toward-mural cell switch of a subpopulation of macrophages with downregulated myeloid cell genes and upregulated mural cell genes, including PDGFRβ. This observation was further strengthened when including unspliced transcripts in the analysis. The macrophage switch was proven functionally relevant, as induction of macrophage-specific PDGFRβ deficiency prevented their perivascular macrophage phenotype, impaired vessel maturation and increased vessel leakiness, which ultimately reduced limb function. In conclusion, macrophages in adult ischemic tissue were demonstrated to undergo a cellular program to morphologically, transcriptomically and functionally resemble mural cells while weakening their macrophage identity. The macrophage-to-mural cell-like phenotypic switch is crucial for restoring tissue function and warrants further exploration as a potential target for immunotherapies to enhance healing., (© 2024. The Author(s).)

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., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

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., Competing Interests: Disclosures None.

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 (IGFBP5 EKO ) facilitated the recovery of blood vessel function and limb necrosis in HLI mice. Moreover, skin damage healing and aortic ring sprouting were faster in IGFBP5 EKO 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 IGFBP5 EKO 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., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

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., Competing Interests: Conflict of interest statement None., (Copyright © 2024 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright: © 2024 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s).)

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.

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.

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., Competing Interests: The authors declare no conflicts of interest in this work., (© 2024 Tang et al.)

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.

We present a two-stage model for the study of chronic hind limb ischemia in rats. In the area of ischemia, sclerotic changes with atrophic rhabdomyocytes and reduced vascularization were revealed. CD31 expression in the endothelium increased proportionally to the number of vessels in the ischemic zone, and at the same time, focal expression of βIII-tubulin was detected in the newly formed nerve fibers. These histological features are equivalent to the development of peripheral arterial disease in humans, which allows using our model in the search for new therapeutic strategies., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Objective: This study aimed to investigate the effect of human adipose tissue derived stromal vascular fraction (AD-SVF) and mesenchymal stem cells (AD-MSCs) on blood flow recovery and neovascularisation in a rat hindlimb ischaemia model., Methods: SVF was isolated using an automated centrifugal system, and AD-MSCs were obtained from adherent cultures of SVF cells. Rats were divided into four groups of six rats each: non-ischaemia (Group 1); saline treated ischaemia (Group 2); SVF treated ischaemia (Group 3); and AD-MSC treated ischaemia (Group 4). Unilateral hindlimb ischaemia was induced in Sprague-Dawley rats via femoral artery ligation. Saline, SVF, or AD-MSCs were injected intramuscularly into the adductor muscle intra-operatively. Cell viability was calculated as the percentage of live cells relative to total cell number. Blood flow improvement, muscle fibre injury, and angiogenic properties were validated using thermal imaging and histological assessment., Results: The viabilities of SVF and AD-MSCs were 83.3% and 96.7%, respectively. Group 1 exhibited no significant temperature difference between hindlimbs, indicating a lack of blood flow changes. The temperature gradient gradually decreased in SVF and AD-MSC treated rats compared with saline treated rats. In addition, only normal muscle fibres with peripherally located nuclei were observed in Group 1. Groups 3 and 4 exhibited significantly fewer centrally located nuclei, indicating less muscle damage compared with Group 2. Regarding angiogenic properties, CD31 staining of endothelial cells showed similar patterns among all groups, whereas expression of vascular endothelial growth factor, as a crucial angiogenesis factor, was enhanced in the SVF and AD-MSC treated groups., Conclusion: SVF and AD-MSCs improved blood flow and neovascularisation in a rat hindlimb ischaemia model, suggesting their potential ability to promote angiogenesis. Further extensive research is warranted to explore their potential applications in the treatment of severe lower extremity arterial disease., (Copyright © 2023. Published by Elsevier B.V.)

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 (K trans ) 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 K trans 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 K trans 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 K trans 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 sum Average ., 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., (© 2024 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Objective: To describe the microvascular anatomy of the equine hind limb suspensory ligament., Animals: 18 hind limbs harvested from 9 adult horses euthanized for reasons unrelated to lameness., Methods: A catheter was placed in the transected cranial tibial artery at the level of the mid-distal tibia for each hind limb and used to inject 120 to 150 mL of contrast medium (2 limbs) to identify principal vasculature using contrast-enhanced CT or India ink (11 limbs) to identify microvasculature using the Spalteholz tissue-clearing technique. Routine histologic evaluation was performed on transverse sections from 4 hind limbs., Results: The hind limb suspensory ligament is principally supplied by branches of the medial and lateral plantar metatarsal arteries and, to a lesser extent, the medial and lateral plantar arteries as well as the associated proximal and distal deep plantar arches. A uniformly distributed intraligamentous microvascular supply was observed without relative deficiencies in vascularity between the proximal, midbody, and distal regions. Histologic examination supported these findings, demonstrating a network of connective tissue surrounding and entering the suspensory ligament containing cross-sections of branches of the principal vasculature., Clinical Relevance: The equine hind limb suspensory ligament has a uniformly distributed and abundant microvascular supply throughout its length, with no evidence of relative deficiency of vascular supply in any region. A region of hypovascularity does not appear to be a viable explanation for the high rate of injury to and commonality of lameness associated with the proximal hind suspensory ligament in horses.

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.

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-β-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.

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 + ., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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.

Critical limb ischemia (CLI) is caused by severe arterial blockage with reduction of blood flow. The aim of this study was to determine whether therapeutic angiogenesis using cellular communication network factor 2 (CCN2) would be useful for treating CLI in an animal model. Recombinant CCN2 was administered intramuscularly to male C57BL/6J mice with hind limb ischemia. The therapeutic effect was evaluated by monitoring blood flow in the ischemic hind limb. In an in vivo assay, CCN2 restored blood flow in the ischemic hind limb by promoting both angiogenesis and lymphangiogenesis. VEGF-A and VEGF-C expression levels increased in the ischemic limb after treatment with CCN2. In an in vitro assay, CCN2 promoted proliferation of vascular and lymphatic endothelial cells, and it upregulated expression of Tgfb1 followed by expression of Vegfc and Vegfr3 in lymphatic endothelial cells under hypoxia. Suppression of Tgfb1 did not affect the activity of CCN2, activation of the TGF-β/SMAD signaling pathway, or expression of Vegfr3 in lymphatic endothelial cells. In summary, treatment using recombinant CCN2 could be a promising therapeutic strategy for CLI., (© 2023. The Author(s).)

Ischemic diseases lead to considerable morbidity and mortality, yet conventional clinical treatment strategies for therapeutic angiogenesis fall short of being impactful. Despite the potential of biomaterials to deliver pro-angiogenic molecules at the infarct site to induce angiogenesis, their efficacy has been impeded by aberrant vascular activation and off-target circulation. Here, we present a semisynthetic low-molecular sulfated chitosan oligosaccharide (SCOS) that efficiently induces therapeutic arteriogenesis with a spontaneous generation of collateral circulation and blood reperfusion in rodent models of hind limb ischemia and myocardial infarction. SCOS elicits anti-inflammatory macrophages' (Mφs') differentiation into perivascular Mφs, which in turn directs artery formation via a cell-to-cell communication rather than secretory factor regulation. SCOS-mediated arteriogenesis requires a canonical Notch signaling pathway in Mφs via the glycosylation of protein O-glucosyltransferases 2, which results in promoting arterial differentiation and tissue repair in ischemia. Thus, this highly bioactive oligosaccharide can be harnessed to direct efficiently therapeutic arteriogenesis and perfusion for the treatment of ischemic diseases.

Ischaemia of the heart and limbs attributable to compromised blood supply is a major cause of mortality and morbidity. The mechanisms of functional angiogenesis remain poorly understood, however. Here we show that FNIP1 plays a critical role in controlling skeletal muscle functional angiogenesis, a process pivotal for muscle revascularization during ischemia. Muscle FNIP1 expression is down-regulated by exercise. Genetic overexpression of FNIP1 in myofiber causes limited angiogenesis in mice, whereas its myofiber-specific ablation markedly promotes the formation of functional blood vessels. Interestingly, the increased muscle angiogenesis is independent of AMPK but due to enhanced macrophage recruitment in FNIP1-depleted muscles. Mechanistically, myofiber FNIP1 deficiency induces PGC-1α to activate chemokine gene transcription, thereby driving macrophage recruitment and muscle angiogenesis program. Furthermore, in a mouse hindlimb ischemia model of peripheral artery disease, the loss of myofiber FNIP1 significantly improved the recovery of blood flow. Thus, these results reveal a pivotal role of FNIP1 as a negative regulator of functional angiogenesis in muscle, offering insight into potential therapeutic strategies for ischemic diseases., (© 2023. The Author(s).)

Hydrogen sulfide (H 2 S) promotes microangiogenesis and revascularization after ischemia. Neovascularization starts with the destruction of intercellular junctions and is accompanied by various endothelial cell angiogenic behaviors. Follistatin-like 1 (FSTL1) is a cardiovascular-protective myokine that works against ischemic injury. The present study examined whether FSTL1 was involved in H 2 S-induced angiogenesis and explored the underlying molecular mechanism. We observed that H 2 S accelerated blood perfusion after ischemia in the mouse hindlimb ischemia model. Western blot analysis showed that H 2 S stabilized FSTL1 transcript and increased FSTL1 and Human antigen R (HuR) levels in skeletal muscle. RNA-interference HuR significantly inhibited the H 2 S-promoted increase in FSTL1 levels. Exogenous FSTL1 promoted the wound-healing migration of human umbilical vein endothelial cells (HUVECs) and increased monolayer endothelial barrier permeability. Immunostaining showed that FSTL1 increased interendothelial gap formation and decreased VE-Cadherin, Occludin, Connexin-43, and Claudin-5 expression. In addition, FSTL1 significantly increased the phosphorylation of Src and VEGFR2. However, the Src inhibitor, not the VEGFR2 inhibitor, could block FSTL1-induced effects in angiogenesis. In conclusion, we demonstrated that H 2 S could upregulate the expression of FSTL1 by increasing the HuR levels in skeletal muscle, and paracrine FSTL1 could initiate angiogenesis by opening intercellular junctions via the Src signaling pathway. NEW & NOTEWORTHY The myocyte-derived paracrine protein FSTL1 acts on vascular endothelial cells and initiates the process of angiogenesis by opening the intercellular junction via activating Src kinase. H 2 S can significantly upregulate FSTL1 protein levels in skeletal muscles by increasing HuR expression.

Despite recent progress in medical and endovascular therapy, the prognosis for patients with critical limb ischemia (CLI) remains poor. In response, various stem cells and growth factors have been assessed for use in therapeutic neovascularization and limb salvage in CLI patients. However, the clinical outcomes of cell-based therapeutic angiogenesis have not provided the promised benefits, reinforcing the need for novel cell-based therapeutic angiogenic strategies to cure untreatable CLI. In the present study, we investigated genetically engineered mesenchymal stem cells (MSCs) derived from human bone marrow that continuously secrete stromal-derived factor-1α (SDF1α-eMSCs) and demonstrated that intramuscular injection of SDF1α-eMSCs can provide long-term paracrine effects in limb ischemia and effectively contribute to vascular regeneration as well as skeletal muscle repair through increased phosphorylation of ERK and Akt within the SDF1α/CXCR4 axis. These results provide compelling evidence that genetically engineered MSCs with SDF-1α can be an effective strategy for successful limb salvage in limb ischemia., (© 2023. The Author(s).)

Peripheral artery disease (PAD) causes an ischemic myopathy contributing to patient disability and mortality. Most preclinical models to date use young, healthy rodents with limited translatability to human disease. Although PAD incidence increases with age, and obesity is a common comorbidity, the pathophysiologic association between these risk factors and PAD myopathy is unknown. Using our murine model of PAD, we sought to elucidate the combined effect of age, diet-induced obesity and chronic hindlimb ischemia (HLI) on (1) mobility, (2) muscle contractility, and markers of muscle (3) mitochondrial content and function, (4) oxidative stress and inflammation, (5) proteolysis, and (6) cytoskeletal damage and fibrosis. Following 16-weeks of high-fat, high-sucrose, or low-fat, low-sucrose feeding, HLI was induced in 18-month-old C57BL/6J mice via the surgical ligation of the left femoral artery at 2 locations. Animals were euthanized 4-weeks post-ligation. Results indicate mice with and without obesity shared certain myopathic changes in response to chronic HLI, including impaired muscle contractility, altered mitochondrial electron transport chain complex content and function, and compromised antioxidant defense mechanisms. However, the extent of mitochondrial dysfunction and oxidative stress was significantly greater in obese ischemic muscle compared to non-obese ischemic muscle. Moreover, functional impediments, such as delayed post-surgical recovery of limb function and reduced 6-minute walking distance, as well as accelerated intramuscular protein breakdown, inflammation, cytoskeletal damage, and fibrosis were only evident in mice with obesity. As these features are consistent with human PAD myopathy, our model could be a valuable tool to test new therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Regenerative therapeutics for treating peripheral arterial disease are an appealing strategy for creating more durable solutions for limb ischemia. In this work, we performed preclinical testing of an injectable formulation of syndecan-4 proteoliposomes combined with growth factors as treatment for peripheral ischemia delivered in an alginate hydrogel. We tested this therapy in an advanced model of hindlimb ischemia in rabbits with diabetes and hyperlipidemia. Our studies demonstrate enhancement in vascularity and new blood vessel growth with treatment with syndecan-4 proteoliposomes in combination with FGF-2 or FGF-2/PDGF-BB. The effects of the treatments were particularly effective in enhancing vascularity in the lower limb with a 2-4 increase in blood vessels in the treatment group in comparison to the control group. In addition, we demonstrate that the syndecan-4 proteoliposomes have stability for at least 28 days when stored at 4°C to allow transport and use in the hospital environment. In addition, we performed toxicity studies in the mice and found no toxic effects even when injected at high concentration. Overall, our studies support that syndecan-4 proteoliposomes markedly enhance the therapeutic potential of growth factors in the context of disease and may be promising therapeutics for inducing vascular regeneration in peripheral ischemia. STATEMENT OF SIGNIFICANCE: Peripheral ischemia is a common condition in which there is a lack of blood flow to the lower limbs. This condition can lead to pain while walking and, in severe cases, critical limb ischemia and limb loss. In this study, we demonstrate the safety and efficacy of a novel injectable therapy for enhancing revascularization in peripheral ischemia using an advanced large animal model of peripheral vascular disease using rabbits with hyperlipidemia and diabetes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The senior author (ABB) has a US patent on the technology in this manuscript., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background Peripheral arterial disease and critical limb ischemia are cardiovascular complications associated with vascular insufficiency, oxidative metabolic dysfunction, and myopathy in the limbs. Estrogen-related receptor gamma (ERRγ) has emerged as a dual regulator of paracrine angiogenesis and oxidative metabolism through transgenic mouse studies. Here our objective was to investigate whether postischemic intramuscular targeting of ERRγ via gene therapy promotes ischemic recovery in a preclinical model of peripheral arterial disease/critical limb ischemia. Methods and Results Adeno-associated virus 9 (AAV9) Esrrg gene delivery vector was developed and first tested via intramuscular injection in murine skeletal muscle. AAV9-Esrrg robustly increased ERRγ protein expression, induced angiogenic and oxidative genes, and boosted capillary density and succinate dehydrogenase oxidative metabolic activity in skeletal muscles of C57Bl/6J mice. Next, hindlimb ischemia was induced via unilateral femoral vessel ligation in mice, followed by intramuscular AAV9-Esrrg (or AAV9-green fluorescent protein) gene delivery 24 hours after injury. ERRγ overexpression increased ischemic neoangiogenesis and markers of endothelial activation, and significantly improved ischemic revascularization measured using laser Doppler flowmetry. Moreover, ERRγ overexpression restored succinate dehydrogenase oxidative metabolic capacity in ischemic muscle, which correlated with increased mitochondrial respiratory complex protein expression. Most importantly, myofiber size to number quantification revealed that AAV9-Esrrg restores myofibrillar size and mitigates ischemia-induced myopathy. Conclusions These results demonstrate that intramuscular AAV9-Esrrg delivery rescues ischemic pathology after hindlimb ischemia, underscoring that Esrrg gene therapy or pharmacological activation could be a promising strategy for the management of peripheral arterial disease/critical limb ischemia.

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1 -/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1 -/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

Background: Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia., Methods: The Actin CreER /R26 VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions., Results: Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105 high Sca1 low , which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105 low Sca1 high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice., Conclusions: These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease., Competing Interests: Disclosures None.

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7 + myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD., Competing Interests: Z-JL and OCV along with the University of Miami hold intellectual property of the E-selectin/AAV gene therapy-based cell modification technologies and have been licensed to Ambulero, Inc., a new start-up company out of the University of Miami that focuses on developing new vascular treatments for ischemic conditions., (Copyright © 2023 Antoine J. Ribieras et al.)

Background: Angiogenesis is a promising strategy for those with peripheral artery disease. Macrophage-centered inflammation is intended to govern the deficiency of the angiogenic response after hindlimb ischemia. However, little is known about the mechanism of macrophage activation beyond signals from cytokines and chemokines. We sought to identify a novel mechanical signal from the ischemic microenvironment that provokes macrophages and the subsequent inflammatory cascade and to investigate the potential role of Piezo-type mechanosensitive ion channels (Piezo) on macrophages during this process., Methods: Myeloid cell-specific Piezo1 (Piezo-type mechanosensitive ion channel component 1) knockout ( Piezo1 ΔMΦ ) mice were generated by crossing Piezo1 fl/fl ( LysM-Cre -/- ; Piezo1 flox/flox ) mice with LysM-Cre transgenic mice to assess the roles of Piezo1 in macrophages after hindlimb ischemia. Furthermore, in vitro studies were carried out in bone marrow-derived macrophages to decipher the underlying mechanism., Results: We found that tissue stiffness gradually increased after hindlimb ischemia, as indicated by Young's modulus. Compared to Piezo2, Piezo1 expression and activation were markedly upregulated in macrophages from ischemic tissues in concurrence with increased tissue stiffness. Piezo1 ΔMΦ mice exhibited improved perfusion recovery by enhancing angiogenesis. Matrigel tube formation assays revealed that Piezo1 deletion promoted angiogenesis by enhancing FGF2 (fibroblast growth factor-2) paracrine signaling in macrophages. Conversely, activation of Piezo1 by increased stiffness or the agonist Yoda1 led to reduced FGF2 production in bone marrow-derived macrophages, which could be blocked by Piezo1 silencing. Mechanistically, Piezo1 mediated extracellular Ca 2+ influx and activated Ca 2+ -dependent CaMKII (calcium/calmodulin-dependent protein kinase II)/ETS1 (ETS proto-oncogene 1) signaling, leading to transcriptional inactivation of FGF2., Conclusions: This study uncovers a crucial role of microenvironmental stiffness in exacerbating the macrophage-dependent deficient angiogenic response. Deletion of macrophage Piezo1 promotes perfusion recovery after hindlimb ischemia through CaMKII/ETS1-mediated transcriptional activation of FGF2. This provides a promising therapeutic strategy to enhance angiogenesis in ischemic diseases.

Aims: Narrowing or occlusion of arteries that supply the limbs can evolve to critical limb ischemia. M-CSF promotes proliferation, differentiation and survival of monocytes and macrophages, and polarization of macrophages to M2-subtype, which are essential elements for vessel formation and tissue repair. Based on these properties of M-CSF, we hypothesize that transfection of M-CSF into ischemic limbs may promote vessel formation and repair of ischemic limbs., Main Methods: Hindlimb ischemia was surgically induced in 10-12 weeks old Balb/c and gene therapy was performed with intramuscular application of either uP-MCSF or uP plasmids (100 μg). Macrophage and monocyte subpopulations were assessed by flow cytometry and blood flow was monitored by Laser Doppler Perfusion Imaging (LDPI). Thirty days after transfection, we assessed gastrocnemius mass and muscle force, subsequently collecting the muscle for histology., Key Findings: We successfully developed the uP-MCSF plasmid, which increases M-CSF expression in the muscle transiently. Thirty days after uP-MCSF gene therapy in ischemic muscles, the treated group presented: improved muscle force, reduced fibrosis and increased arteriogenesis, although LDPI analysis did not show any significant difference in blood flow among groups. Noteworthy, we observed a temporary increase in MHCII high CD206 high macrophages after uP-MCSF transfection., Significance: M-CSF gene therapy improved ischemic muscle functionality by promoting arteriogenesis and decreasing fibrosis, likely through increased MHCII high CD206 high macrophages and not via classically known M2-macrophages., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background Severe hindlimb ischemia is a chronic disease with poor prognosis that can lead to amputation or even death. This study aimed to assess the therapeutic effect of liraglutide on hind-limb ischemia in type 2 diabetic mice and to elucidate the underlying mechanism. Methods and Results Blood flow reperfusion and capillary densities after treatment with liraglutide or vehicle were evaluated in a mouse model of lower-limb ischemia in a normal background or a background of streptozotocin-induced diabetes. The proliferation, migration, and tube formation of human umbilical vein endothelial cells were analyzed in vitro upon treatment with liraglutide under normal-glucose and high-glucose conditions. Levels of phospho-Akt, phospho-endothelial nitric oxide synthase, and phospho-extracellular signal-related kinases 1 and 2 under different conditions in human umbilical vein endothelial cells and in ischemic muscle were determined by western blotting. Liraglutide significantly improved perfusion recovery and capillary density in both nondiabetic and diabetic mice. Liraglutide also promoted, in a concentration-dependent manner, the proliferation, migration, and tube formation of normal glucose- and high glucose-treated human umbilical vein endothelial cells, as well as the phosphorylation of Akt, endothelial nitric oxide synthase, and extracellular signal-related kinases 1 and 2 both in vitro and in vivo. The liraglutide antagonist exendin (9-39) reversed the promoting effects of liraglutide on human umbilical vein endothelial cell functions. Furthermore, exendin (9-39), LY294002, and PD98059 blocked the liraglutide-induced activation of Akt/endothelial nitric oxide synthase and extracellular signal-related kinases 1 and 2 signaling pathways. Conclusions These studies identified a novel role of liraglutide in modulating ischemia-induced angiogenesis, possibly through effects on endothelial cell function and activation of Akt/endothelial nitric oxide synthase and extracellular signal-related kinases 1 and 2 signaling, and suggested the glucagon-like peptide-1 receptor may be an important therapeutic target in diabetic hind-limb ischemia.

Ischemia-reperfusion (I/R) injury is a complication impacting multiple organs and tissues in clinical conditions ranging from peripheral arterial disease to musculoskeletal trauma and myocardial infarction. Stem cell-derived extracellular vesicles (EVs) may represent one therapeutic resource for preventing the tissue damage associated with I/R injury. Here we tested the hypothesis that lyophilized extracellular vesicles derived from adipose stem cells could serve as an "off-the-shelf" treatment modality for I/R injury in a mouse hindlimb ischemia model. Ischemia was induced for 90 min using a rubber band tourniquet and extracellular vesicles (0, 50, or 100 µg) administered via tail vein injection immediately prior to reperfusion. Perfusion was measured prior to, during, and after ischemia using laser Doppler imaging. Serum and tissue were collected 24 h after reperfusion. Mass spectrometry (MS)-based proteomics was used to characterize the EV cargo and proteins from the ischemic and non-ischemic hindlimb. Inflammatory cytokines were measured in muscle and serum using a multiplex array. Results indicate that EVs significantly increase reperfusion and significantly increase expression of the anti-inflammatory factor annexin a1 in skeletal muscle; however, the increased reperfusion was also associated with a marked decrease in muscle structural proteins such as dystrophin, plectin, and obscurin. Circulating inflammatory cytokines TNF-alpha and IL-6 were increased with EV treatment, and serum TNF-alpha showed a significant, positive correlation with reperfusion level. These findings suggest that, while EVs may enhance reperfusion, the increased reperfusion can negatively impact muscle tissue and possibly remote organs. Alternative approaches, such as targeting mitochondrial permeability, may be more effective at mitigating I/R injury.

In this research, we introduced a novel strategy for fabricating cell sheets (CSs) prepared by simply adding a fibrinogen solution to growth medium without using any synthetic polymers or chemical agents. We confirmed that the fibrinogen-based CS could be modified for target tissue regardless of size, shape, and cell types. Also, fibrinogen-based CSs were versatile and could be used to form three-dimensional (3D) CSs such as multi-layered CSs and those mimicking native blood vessels. We also prepared fibrinogen-based spheroid sheets for the treatment of ischemic disease. The fibrinogen-based spheroid sheets had much higher in vitro tubule formation and released more angiogenic factors compared to other types of platform in this research. We transplanted fibrinogen-based spheroid sheets into a mouse hindlimb ischemia model and found that fibrinogen-based spheroid sheets showed significantly improved physiological function and blood perfusion rates compared to the other types of platform in this research., (© 2023 IOP Publishing Ltd.)