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

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

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

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

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

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

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

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

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

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

Sulfated oligosaccharide activates endothelial Notch for inducing macrophage-associated arteriogenesis to treat ischemic diseases.

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.

FNIP1 abrogation promotes functional revascularization of ischemic skeletal muscle by driving macrophage recruitment.

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.

Rescue of murine hind limb ischemia via angiogenesis and lymphangiogenesis promoted by cellular communication network factor 2.

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-ß/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.

Vascular regeneration and skeletal muscle repair induced by long-term exposure to SDF-1α derived from engineered mesenchymal stem cells after hindlimb ischemia.

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.

SUV39H1 Inhibits Angiogenesis in Limb Ischemia of Mice.

Peripheral arterial disease (PAD), characterized by atherosclerosis of the peripheral arteries or even amputation, has threatened public life and health. However, the underlying mechanism remains largely obscure. SUV39H1, a histone methyltransferase, could specifically methylate lysine 9 of histone H3 and act as a repressor in transcriptional activity. The study aimed to investigate the role of SUV39H1 in limb ischemia. C57BL/6 male mice were randomly divided into Sham or Model groups to investigate the expression of SUV39H1 in the ischemic limbs. Then, pharmaceutical inhibition or genetic deletion of SUV39H1 in the limb ischemia mice model was performed to confirm its effect on limb ischemia. The blood perfusion was quantified by laser speckle contrast imaging (LSCI). Capillary density and muscle edema were measured by CD31 immunohistochemical staining and HE staining. The expressions of SUV39H1 and Catalase were confirmed by western blot. Transcriptome sequencing of siSUV39H1 in human umbilical vein endothelial cells (HUVECs) was used to explore the regulation mechanism of SUV39H1 on angiogenesis. The results showed that SUV39H1 was highly expressed in the ischemic muscle tissue of the mice. Pharmaceutical inhibition or genetic deletion of SUV39H1 significantly improved blood perfusion, capillary density, and angiogenesis in ischemic muscle tissue. Cell experiments showed that SUV39H1 knockdown promoted cell migration, tube formation, and mitochondrial membrane potential in endothelial cells under oxidative stress. The transcriptome sequencing results unmasked mechanisms of the regulation of angiogenesis induced by SUV39H1. Finally, Salvianolic acid B and Astragaloside IV were identified as potential drug candidates for the improvement of endothelial function by repressing SUV39H1. Our study reveals a new mechanism in limb ischemia. Targeting SUV39H1 could improve endothelial dysfunction and thus prevent limb ischemia.

Estrogen-Related Receptor Gamma Gene Therapy Promotes Therapeutic Angiogenesis and Muscle Recovery in Preclinical Model of PAD.

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.

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.

Syndecan-4 proteoliposomes enhance revascularization in a rabbit hind limb ischemia model of peripheral ischemia.

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.

Endothelial Progenitor Cells and Macrophage Subsets Recruitment in Postischemic Mouse Hind Limbs.

INTRODUCTION: Peripheral arterial disease (PAD) occurs from atherosclerotic obstruction of arteries in the lower extremities. Restoration of perfusion requires angiogenesis and arteriogenesis through migration and differentiation of endothelial progenitor cells (EPCs) and macrophages at the site of injury. The time of recruitment has not been fully investigated. In this study, we investigated the infiltration of these cells in murine hind limb ischemia (HLI) model of PAD. METHODS: EPCs and M1-like and M2-like macrophages from ischemic skeletal muscles were quantified by flow cytometry at day-0, 1, 3, 7, and 14 post-HLI. RESULTS: The abundance of EPCs increased from day 1 and was highest on day 7 until day 14. M1-like population similarly increased and was highest on day 14 during the experiment. M2-like population was significantly greater than M1-like at baseline but surpassed the highest value of M1-like by day 7 during the experiment. Muscle regeneration and capillary density also increased and were highest at days 3 and 7, respectively, during the experiment. All mice achieved near full perfusion recovery by day 14. CONCLUSION: Thus, we observed a gradual increase in the percentage of EPC's and this was temporally paralleled with initial increase in M1-like followed by sustained increased in M2-like macrophages and perfusion recovered post-HLI.

E-Selectin/AAV Gene Therapy Promotes Myogenesis and Skeletal Muscle Recovery in a Mouse Hindlimb Ischemia Model.

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.

Diet-induced obesity augments ischemic myopathy and functional decline in a murine model of peripheral artery disease.

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.

A Combination of Distinct Vascular Stem/Progenitor Cells for Neovascularization and Ischemic Rescue.

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 ActinCreER/R26VT2/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+CD105highSca1low, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high 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.

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions.

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.

Macrophages mobilized by the overexpression of the macrophage-colony stimulating factor promote efficient recovery of the ischemic muscle functionality.

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 MHCIIhighCD206high macrophages after uP-MCSF transfection. SIGNIFICANCE: M-CSF gene therapy improved ischemic muscle functionality by promoting arteriogenesis and decreasing fibrosis, likely through increased MHCIIhighCD206high macrophages and not via classically known M2-macrophages.

Lyophilized Extracellular Vesicles from Adipose-Derived Stem Cells Increase Muscle Reperfusion but Degrade Muscle Structural Proteins in a Mouse Model of Hindlimb Ischemia-Reperfusion Injury.

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.