Gangrene, revascularization, and limb function improved with E-selectin/adeno-associated virus gene therapy

Peripheral arterial disease (PAD) affects more than 200 million people worldwide and is caused by occlusions resulting from atherosclerosis in arteries supplying blood to the lower limbs. Treatments for limb ischaemia in PAD is an unmet medical need. Novel treatments are sought to improve limb blood supply and function in PAD patients unlikely to benefit from existing treatments such as surgical revascularisation, structured exercise programs and cilostozol. Currently, the main pre-clinical experimental model employed in PAD research is based on induction of acute hind limb ischemia (HLI) which results in rapid natural recovery of blood supply to ischaemic tissues. There are concerns regarding the ability to translate findings from this mouse model to PAD patients and a clinically relevant mouse model of HLI is lacking. Evidence suggests metformin may be a potential treatment for PAD and angiotensin converting enzyme 2 (ACE2) may play a role in limb ischaemia. This thesis aimed to develop a clinically relevant mouse model of PAD that involved a two-stage induction of HLI and examine the effect of exercise training, metformin and ACE2 deficiency in the two-stage model of HLI. Two-stage HLI was induced in male Apolipoprotein E (ApoE-/-) deficient mice by slow onset of severe ischemia over 14 days. This 2-stage HLI model was compared to the acute HLI model and sham controls. Limb blood supply was assessed by Laser Doppler Perfusion Imaging (LDI). Ambulatory ability was assessed using a treadmill test and established scoring scales. Running wheel exercise training was examined in the two-stage mouse model and limb function was assessed by a treadmill exercise test and blood supply was assessed by LDI. Next, the effect of metformin on limb ischaemia was assessed by LDI in the two-stage HLI mouse model. Lastly, the effect of ACE2 deficiency (ACE2-/y) on limb ischaemia was examined in the two-stage mouse model of HLI. HLI was significantly more severe in mice receiving the two-stage compared to the acute HLI induction procedure as assessed by LDI (p=0.013), and reflected a higher ischemic score (p=0.003) and lower average distance travelled on a treadmill exercise test (p=0.045). Mice with two-stage HLI receiving exercise training showed significantly greater improvement in their ambulatory ability on a treadmill test than the sedentary control group (p=0.003). However, limb blood supply was comparable between mice receiving exercise compared to controls (p=0.700). Mice with two-stage HLI administered metformin had greater blood supply than mice with two-stage HLI receiving vehicle control (p<0.001). The two-stage HLI mice receiving metformin had increased adenosine monophosphate kinase alpha (AMPKα) activity (p=0.041), endothelial nitric oxide synthase (e-NOS) activity (p=0.031), nitric oxide (NO) bioavailability (p=0.024), peroxisome proliferator activated receptor 1 alpha (PGC1-α) expression (p=0.026) and decreased thioredoxin interacting protein (TXNIP) expression (p=0.038) compared to mice with two-stage HLI receiving vehicle. ACE2-/y in mice with two-stage HLI had comparable limb ischaemia to ACE2 unmodified control mice with two-stage HLI (p=0.263). In conclusion, this thesis showed that the novel two-stage mouse model of HLI had severe ongoing HLI and functional impairment. Exercise training augmented treadmill walking capacity in the new mouse model which was independent of changes to limb blood supply and mirroring patient response to exercise therapy. Metformin administration improved limb blood supply in the two-stage mouse model of ongoing HLI. Improvement in blood supply was associated with the upregulation of AMPKα activity, increased activation of e-NOS in the ischaemic muscles, increased bioavailability of circulating NO, increased expression of PGC1α and reduced expression of TXNIP. This suggests metformin may have potential to be used as a treatment to improve limb blood supply in PAD patients. ACE2-/ did not worsen limb blood supply after two-stage HLI induction suggesting ACE2 may not play an important role in limb ischemia. The results do not encourage the pursuit of ACE2 for pharmacological management of PAD in clinical trials and ACE2 is unlikely to be an important target for improving limb blood supply or function in patients with PAD.

Chloroquine improves the response to ischemic muscle injury and increases HMGB1 after arterial ligation

The RGD-modified self-assembling D-form peptide hydrogel enhances the therapeutic effects of mesenchymal stem cells (MSC) for hindlimb ischemia by promoting angiogenesis

Introduction: Peripheral vascular disease affects over ten million Americans. Hypoxia in ischemic limbs typically initiates angiogenic and inflammatory factors to promote angiogenesis in attempt to restore perfusion. Little is known about the inflammatory nature of angiogenesis particularly with respect to anti-inflammatory factors. Interleukin-19 is a uniquely anti-inflammatory Th2 cytokine that promotes angiogenesis in cellular assays. We hypothesized that IL-19 could drive angiogenesis in vivo; the purpose of this study is to characterize a role for IL-19 in restoration of blood flow in the hind-limb ischemia model, and define potential mechanisms. Methods and Results: Three complimentary experiments were designed to clarify IL-19’s angiogenic role. 1- C57BL/6 (n=12) were subject to induced hindlimb ischemia by femoral artery ligation and subject to daily i.p. injections of recombinant mouse IL-19 (10ng/g/day) or PBS for 14 days. Mice were subsequently imaged using Laser Doppler Perfusion Imaging (LDPI). Perfusion index was evaluated by comparing values between ligated and unligated contralateral limbs. C57BL/6 mice injected with rIL-19 showed significantly increased levels of perfusion when compared to PBS injected age-matched cohorts (p&lt;0.01 at day 7, &lt;0.05 at day 10). 2- hindlimb ischemia was similarly induced in both C57BL/6 and IL-19-/- mice on a C57BL/6 background. LDPI values are significantly decreased in IL-19-/- mice when compared to C57BL/6 mice (p&lt;0.05 at day 7). 3- IL-19-/- mice subject to limb ischemia were given daily i.p. injections of rIL-19 or PBS. IL-19-/- mice administered rIL-19 trended (P=0.058) higher perfusion when compared to PBS injected control mice. Immunohistochemistry using CD31 antibody on adductor muscle recovered from these mice showed significantly increased capillary density in IL-19 treated mice compared with controls (P&lt;0.05). cDNA microarray analysis of human microvascular endothelial cells treated with IL-19 determined increased expression of angiogenic cytokines, which was verified by qRT-PCR and western blot. Conclusions: This study is the first to implicate IL-19 as a novel pro-angiogenic cytokine with potential to promote neovascularization and restore blood flow in ischemic tissue.

Fucoidan improves bioactivity and vasculogenic potential of mesenchymal stem cells in murine hind limb ischemia associated with chronic kidney disease

In this issue of Circulation , Baumgartner et al1 ). The plasmid DNA was injected directly into the muscle of ischemic limbs. Anatomic and functional efficacy was demonstrated by increased serum levels of VEGF, improved hemodynamic measurements and angiographic evaluation, reduced pain, increased healing of ischemic ulcers, limb salvage, and immunohistochemical evidence of proliferating endothelial cells in tissue specimens. The authors emphasize that this is the first medical therapy to achieve an increase in limb perfusion that is equivalent to or greater than successful surgical or percutaneous intervention. Direct intramuscular gene transfer of plasmid DNA appears to effectively stimulate collateral vessel growth, despite the lower transfection efficiency that is usually associated with gene therapy in the absence of a viral vector. This result has implications for other clinical trials of gene therapy that use intramuscular naked DNA. The fact that Isner’s group could prepare the plasmid for human use in a university medical center laboratory dedicated to this purpose reveals why gene therapy has moved so rapidly from the laboratory to the clinic, in contrast to protein therapy, which requires expensive manufacturing facilities and years of scale-up effort. Although the plasmid was injected into muscle of the ischemic limb, VEGF levels were apparently elevated in the whole circulation, as evidenced by transient peaks of VEGF in the serum and by edema of the ischemic limb and in some patients, in the opposite limb. The increased collateral vessels, however, are localized to the ischemic limb and do not develop in other areas of the body. This may reflect in part the short half-life of VEGF in the circulation (minutes) as well as the upregulation of receptors for VEGF in ischemic tissue. …

Vascular endothelial growth factor-121 (VEGF121), an angiogenic protein secreted in response to hypoxic stress, binds to VEGF receptors (VEGFRs) overexpressed on vessels of ischemic tissue. The purpose of this study was to evaluate 64Cu-VEGF121 positron emission tomography for noninvasive spatial, temporal, and quantitative monitoring of VEGFR2 expression in a murine model of hindlimb ischemia with and without treadmill exercise training. 64Cu-labeled VEGF121 and a VEGF mutant were tested for VEGFR2 binding specificity in cell culture. Mice (n=58) underwent unilateral ligation of the femoral artery, and postoperative tissue ischemia was assessed with laser Doppler imaging. Longitudinal VEGFR2 expression in exercised and nonexercised mice was quantified with 64Cu-VEGF121 positron emission tomography at postoperative day 8, 15, 22, and 29 and correlated with postmortem gamma-counting. Hindlimbs were excised for immunohistochemistry, Western blotting, and microvessel density measurements. Compared with the VEGF mutant, VEGF121 showed specific binding to VEGFR2. Perfusion in ischemic hindlimbs fell to 9% of contralateral hindlimb on postoperative day 1 and recovered to 82% on day 29. 64Cu-VEGF121 uptake in ischemic hindlimbs increased significantly (P < 0.001) from a control level of 0.61+/-0.17% ID/g (percentage of injected dose per gram) to 1.62+/-0.35% ID/g at postoperative day 8, gradually decreased over the following 3 weeks (0.59+/-0.14% ID/g at day 29), and correlated with gamma-counting (R2 = 0.99). Compared with nonexercised mice, 64Cu-VEGF121 uptake was increased significantly (P < or = 0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R2 = 0.76). Ischemic hindlimb tissue stained positively for VEGFR2. In exercised mice, microvessel density was increased significantly (P<0.001) compared with nonexercised mice. 64Cu-VEGF121 positron emission tomography allows longitudinal spatial and quantitative monitoring of VEGFR2 expression in murine hindlimb ischemia and indirectly visualizes enhanced angiogenesis stimulated by treadmill exercise training.

Hypoxia preconditioning enhances the paracrine abilities of mesenchymal stem cells (MSCs) for vascular regeneration and tissue healing. Implantation of hypoxia-induced mesenchymal stem cells (hi-MSCs) may further improve limb perfusion in a murine model of hindlimb ischemia. This study is aimed at determining whether implantation of hi-MSCs is an effective modality for improving outcomes of treatment of ischemic artery diseases. We evaluated the effects of human bone marrow-derived MSC implantation on limb blood flow in an ischemic hindlimb model. hi-MSCs were prepared by cell culture under 1% oxygen for 24 hours prior to implantation. A total of 1 × 105 MSCs and hi-MSCs and phosphate-buffered saline (PBS) were intramuscularly implanted into ischemic muscles at 36 hours after surgery. Restoration of blood flow and muscle perfusion was evaluated by laser Doppler perfusion imaging. Blood perfusion recovery, enhanced vessel densities, and improvement of function of the ischemia limb were significantly greater in the hi-MSC group than in the MSC or PBS group. Immunochemistry revealed that hi-MSCs had higher expression levels of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor A than those in MSCs. In addition, an endothelial cell-inducing medium showed high expression levels of vascular endothelial growth factor, platelet endothelial cell adhesion molecule-1, and von Willebrand factor in hi-MSCs compared to those in MSCs. These findings suggest that pretreatment of MSCs with a hypoxia condition and implantation of hi-MSCs advances neovascularization capability with enhanced therapeutic angiogenic effects in a murine hindlimb ischemia model.

Background:Peripheral artery disease (PAD) and its severe form, chronic limb-threatening ischemia (CLTI), significantly impair blood flow to the lower extremities, affecting millions of adults globally. Intramuscular adipose tissue (IMAT) and fibrosis accumulation distinguish patients with CLTI from those with mild PAD, suggesting a role in CLTI pathobiology. However, the functional consequences of IMAT in CLTI remain unclear.Methods:We compared gastrocnemius muscle samples from patients with PAD/CLTI, intermittent claudication, and non-PAD individuals. We analyzed bulk RNA sequencing, proteomic, lipidomic, and single-cell/nucleus RNA sequencing datasets. Additionally, we used murine models of hindlimb ischemia (HLI) with genetic manipulation of Pparγ, a key adipogenic transcription factor, specifically in fibro-adipogenic progenitor cells (FAPs), the cellular source of IMAT, to modulate IMAT formation and assessed the impact on limb function and pathology.Results:Patients with CLTI exhibited significantly elevated expression of adipogenic genes and proteins in muscle specimens when compared to non-PAD controls. Murine models showed that increasing IMAT formation significantly worsened ischemic limb muscle strength and work output. In contrast, preventing IMAT formation significantly improved ischemic limb muscle strength and work output. These findings were consistent across both male and female mice, although females had greater tendency to form IMAT compared with male mice.Conclusions:IMAT accumulation is a key determinant of limb function in PAD/CLTI. Our studies demonstrate that targeting IMAT formation could improve limb function in mice with experimental PAD. Together, these findings suggest that developing strategies to limit or reduce IMAT may improve limb function and walking performance in patients with PAD/CLTI, providing a novel therapeutic avenue to address a critical unmet need.

Microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E2 (PGE2) induces angiogenesis through the prostaglandin E2 receptor (EP1-4). Among immune cells, regulatory T cells (Tregs), which inhibit immune responses, have been implicated in angiogenesis, and PGE2 is known to modulate the function and differentiation of Tregs. We hypothesized that mPGES-1/PGE2-EP signalling could contribute to recovery from ischaemic conditions by promoting the accumulation of Tregs. Wild-type (WT), mPGES-1-deficient (mPges-1-/-), and EP4 receptor-deficient (Ep4-/-) male mice, 6-8 weeks old, were used. Hindlimb ischaemia was induced by femoral artery ligation. Recovery from ischaemia was suppressed in mPges-1-/- mice and compared with WT mice. The number of accumulated forkhead box protein P3 (FoxP3)+ cells in ischaemic muscle tissue was decreased in mPges-1-/- mice compared with that in WT mice. Expression levels of transforming growth factor-β (TGF-β) and stromal cell derived factor-1 (SDF-1) in ischaemic tissue were also suppressed in mPges-1-/- mice. The number of accumulated FoxP3+ cells and blood flow recovery were suppressed when Tregs were depleted by injecting antibody against folate receptor 4 in WT mice but not in mPges-1-/- mice. Recovery from ischaemia was significantly suppressed in Ep4-/- mice compared with that in WT mice. Furthermore, mRNA levels of Foxp3 and Tgf-β were suppressed in Ep4-/- mice. Moreover, the number of accumulated FoxP3+ cells in ischaemic tissue was diminished in Ep4-/- mice compared with that in Ep4+/+ mice. These findings suggested that mPGES-1/PGE2 induced neovascularization from ischaemia via EP4 by promoting the accumulation of Tregs. Highly selective EP4 agonists could be useful for the treatment of peripheral artery disease.

Background and aims: Lackluster results from recently completed gene therapy clinical trials of VEGF-A delivered by viral vectors have heightened the need to develop alternative delivery strategies. This study aims to demonstrate the preclinical efficacy and safety of extracellular vesicles (EVs) loaded with VEGF-A mRNA for the treatment of ischemic vascular disease. Methods: After encapsulation of full-length VEGF-A mRNA into fibroblast-derived EVs via cellular nanoporation (CNP), collected VEGF-A EVs were delivered into mouse models ischemic injury. Target tissue delivery was verified by in situ analysis of protein and gene expression. Functional rescue was confirmed by in vivo imaging and histology. The safety of single and serial delivery was demonstrated using immune-based assays. Results: VEGF-A EVs were generated with high mRNA content using a CNP methodology. VEGF-A EV administration demonstrated expression of exogenous VEGF-A mRNA by in situ RNA hybridization and elevated protein expression by Western blot, microscopy, and ELISA. Mice treated with human VEGF-A EVs after femoral or coronary artery ligation exhibited heightened neovascularization in ischemic tissues with increased arterial perfusion and improvement in left ventricular function, respectively. Serial delivery of VEGF-EVs in injured skin showed improved wound healing with repeat administration. Importantly, as compared with AAV and LNP VEGF-A gene therapy modalities, murine VEGF-A EV delivery did not trigger innate or adaptive immune responses at the injection site or systemically. Conclusions: This study demonstrated that VEGF-A EV therapy offers efficient, dose-dependent VEGF-A protein formation with low immunogenicity, resulting in new vessel formation in murine models of ischemic vascular disease.

Mesenchymal stem cells (MSCs) are promising candidates for stem cell-based therapy in ischemic diseases. However, ischemic injury induces pathophysiological conditions, such as oxidative stress and inflammation, which diminish therapeutic efficacy of MSC-based therapy by reducing survival and functionality of transplanted MSCs. To overcome this problem, we explored the effects of melatonin on the proliferation, resistance to oxidative stress, and immunomodulatory properties of MSCs. Treatment with melatonin enhanced MSC proliferation and self-renewal via upregulation of cellular prion protein (PrPC ) expression. Melatonin diminished the extent of MSC apoptosis in oxidative stress conditions by regulating the levels of apoptosis-associated proteins, such as BCL-2, BAX, PARP-1, and caspase-3, in a PrPC -dependent manner. In addition, melatonin regulated the immunomodulatory effects of MSCs via the PrPC -IDO axis. In a murine hind-limb ischemia model, melatonin-stimulated MSCs improved the blood flow perfusion, limb salvage, and vessel regeneration by lowering the extent of apoptosis of affected local cells and transplanted MSCs as well as by reducing infiltration of macrophages. These melatonin-mediated therapeutic effects were inhibited by silencing of PrPC expression. Our findings for the first time indicate that melatonin promotes MSC functionality and enhances MSC-mediated neovascularization in ischemic tissues through the upregulation of PrPC expression. In conclusion, melatonin-treated MSCs could provide a therapeutic strategy for vessel regeneration in ischemic disease, and the targeting of PrPC levels may prove instrumental for MSC-based therapies.

BackgroundThe proangiogenic capacity of adipose tissue and its derivatives has been demonstrated in a variety of studies. The paracrine mechanism of the cellular component is considered to play a critical role in the regenerative properties of these tissues. However, cell-based therapy for clinical application has been hindered by limitations such as safety, immunogenicity issues, and difficulties in cell preservation, transportation, and phenotype control. In the current study, we aimed to produce a cell-free extract directly from human fat tissue and evaluate its potential therapeutic efficacy.MethodsWe developed a novel physical approach to produce a cell-free aqueous extract from human fat tissue (fat extract (FE)). The therapeutic potential of FE was investigated in the ischemic hindlimb model of nude mice. After establishment of hindlimb ischemia with ligation of the left femoral artery and intramuscular injection of FE, blood perfusion was monitored at days 0, 7, 14, 21, and 28. Tissue necrosis and capillary density were evaluated. Enzyme-linked immunosorbent assay was used to analyze the growth factors contained in FE. Moreover, the proliferation, migration, and tube formation ability were tested on human umbilical vein endothelial cells (HUVECs) in vitro when treated with FE. The proangiogenic ability of FE was further assessed in an in-vivo Matrigel plug assay.ResultsFE was prepared and characterized. The intramuscular injection of FE into the ischemic hindlimb of mice attenuated severe limb loss and increased blood flow and capillary density of the ischemic tissue. Enzyme-linked immunosorbent assay showed that FE contained high levels of various growth factors. When added as a cell culture supplement, FE promoted HUVEC proliferation, migration, and tube formation ability in a dose-dependent manner. The subcutaneous injection of Matrigel infused with FE enhanced vascular formation.ConclusionsWe developed a novel cell-free therapeutic agent, FE, produced from human adipose tissue. FE was able to attenuate ischemic injury and stimulate angiogenesis in ischemic tissues. This study indicates that FE may represent a novel cell-free therapeutic agent in the treatment of ischemic disorders.

Background: Although clock genes generate oscillations in about 10 % of all gene expressions as a 24-hour cycle in the cellular level cooperating with the central clock, little is known about the role of clock genes in angiogenesis. Objective: The aim of this study was to determine if a peripheral clock would modulate angiogenesis in a hind limb ischemia (HLI) model. Methods and Results: First, we surgically induced the HLI model in C57BL/6J (wild type; WT) mice (N=6 for each time point) and checked mRNA expressions of clock genes in a local ischemic tissue by PCR array in the setting of blood perfusion recovery. In vitro study, we next investigated those gene expressions in human umbilical vein endothelial cells (HUVECs) with or without stimulation by growth factors. Those data demonstrated that the peripheral clock genes were modulated in ECs and tissues in the presence of growth factors and tissue ischemia. Then, we examined whether inhibition of clock gene expressions had any effects on angiogenesis. For this study, we focused on Cryptochrome (Cry), which is well known as one of the core-loop forming clock genes producing circadian rhythm in mammals. Our loss-of-function study revealed that the abilities of proliferation, migration and tube formation were significantly inhibited by Cry1 and Cry2 double knockdown in HUVECs. Interestingly, although the knockdown of Cry1 and Cry2 changed the mRNA expression of Period2, it did not affect those of BMAL1 and Clock in HUVECs. Finally, we tested if Cry1 and Cry2 double hetero knockout mice of HLI models displayed worse blood perfusion recoveries with deterioration of angiogenesis. As results, compared with control WT mice, Cry1 and Cry2 double knockout mice showed the low capillary density detected by CD31-immunohistochemistry and low tissue blood perfusion demonstrated by laser Doppler perfusion imaging (LDPI) in a HLI. Conclusion: Our data indicated that Cryptochrome as a peripheral clock plays an important role in angiogenesis of the reparative tissue ischemia model.

Viral vector systems are crucial in delivering therapeutic genetic material for gene and cell therapy in clinical settings. With new progress in viral vector-based gene therapies, healthcare professionals and pharmacists must stay informed and apply appropriate biosafety precautions when handling these products. Comprehensive guidelines covering biosafety measures for every viral vector-based gene therapy, including storage, preparation, administration, and disposal, remain scarce. As viral vector-based gene therapy advances rapidly, it is critical to equip healthcare professionals with the expertise to safely manage these agents in a clinical setting. As gene therapies become more integrated into mainstream medicine, with 43 cell and gene therapy products approved as of 2025, pharmacists, nurses, and caregivers face new safety challenges when handling these therapies. While guidelines such as United States Pharmacopeia (USP) 797 and USP 800 provide a foundation for handling viral vector-based gene therapies, no single resource consolidates all biosafety considerations for healthcare professionals. This review provides an overview of the viral vector-based gene therapy landscape. It compiles best practices from various sources to establish a potential standardized approach to guide safe handling procedures for viral vector systems in a healthcare setting. By adopting the biosafety strategies outlined in this article, healthcare staff and pharmacists can safely manage viral vectors while developing standard operating procedures tailored to their specific clinical environments. Implementing these measures will support the continued advancement of gene therapy while ensuring the safety of healthcare providers, patients, and the environment.