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Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood–brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.

Background Post-surgical lymphedema frequently occurs following lymph node dissection. The murine tail is the most commonly used model to study secondary lymphedema. The murine hindlimb model offers a more clinically translatable approach but results in the literature have been inconsistent. The purpose of this study is to optimize the murine hindlimb lymphedema to achieve consistent results and assess the utility of radiation. Methods C57BL/6 mice underwent either 20-Gy irradiation of one hindlimb 7 days prior to surgery ( n  = 11) or no radiation ( n  = 9). For all mice, a circumferential skin incision was created at the proximal hindlimb. Lymphatics were identified and disrupted. Popliteal lymph nodes were excised. Paw thickness was measured and near-infrared laser lymphangiography was used to assess lymphatic function. Results The average paw thickness of the operated hindlimb in irradiated mice on postoperative day (POD) 14 was 3.5 ± 0.3 cm compared to 2.1 ± 0.05 cm on the contralateral limb ( p  = 0.0001). Lymphangiography on POD-42 showed significantly worse lymphatic function in the operated hindlimb compared to the control hindlimb ( p  = 0.003). For the non-radiated mice, the paw thickness was 2.5 ± 0.2 cm on POD-42 compared to the contralateral limb (2.1 ± 0.1 cm) ( p  = 0.0002) but smaller than radiated hindlimb group (3.2 ± 0.1 cm) ( p  = 0.0002). The nonradiated mice had significantly greater paw thickness than the control limb until POD-56 whereas the radiated mice sustained significant paw thickness to POD-90. Conclusion Radiation of the murine hindlimb model results in sustained lymphedema compared to non-irradiated mice. The murine hindlimb lymphedema model is clinically more translatable than the murine tail model with consistent results.

Angiogenesis induction into damaged sites has long been an unresolved issue. Local treatment with pro-angiogenic molecules has been the most common approach. However, this approach has critical side effects including inflammatory coupling, tumorous vascular activation, and off-target circulation. Here, the concept that a structure can guide desirable biological function is applied to physically engineer three-dimensional channel networks in implant sites, without any therapeutic treatment. Microchannel networks are generated in a gelatin hydrogel to overcome the diffusion limit of nutrients and oxygen three-dimensionally. Hydrogel implantation in mouse and porcine models of hindlimb ischemia rescues severely damaged tissues by the ingrowth of neighboring host vessels with microchannel perfusion. This effect is guided by microchannel size-specific regenerative macrophage polarization with the consequent functional recovery of endothelial cells. Multiple-site implantation reveals hypoxia and neighboring vessels as major causative factors of the beneficial function. This technique may contribute to the development of therapeutics for hypoxia/inflammatory-related diseases. Restoration of blood flow to damaged sites has commonly involved treatment with pro-angiogenic molecules but these have undesired side effects. Here the authors present a microchannel-patterned gelatin hydrogel that is able to rescue mouse and porcine models of hindlimb ischemia.

Mouse hind limb ischemia is the most common used preclinical model for peripheral arterial disease and critical limb ischemia. This model is used to investigate the mechanisms of neovascularization and to develop new therapeutic agents. The literature shows many variations in the model, including the method of occlusion, the number of occlusions, and the position at which the occlusions are made to induce hind limb ischemia. Furthermore, predefined end points and the histopathological and radiological analysis vary. These differences hamper the correlation of results between different studies. In this review, variations in surgical methods of inducing hind limb ischemia in mice are described, and the consequences of these variations on perfusion restoration and vascular remodeling are discussed. This study aims at providing the reader with a comprehensive overview of the methods so far described, and proposing uniformity in research of hind limb ischemia in a mouse model.

Chronic kidney disease (CKD) substantially increases the severity of peripheral arterial disease (PAD) symptomology, however, the biological mechanisms remain unclear. The objective herein was to determine the impact of CKD on PAD pathology in mice. C57BL6/J mice were subjected to a diet-induced model of CKD by delivery of adenine for six weeks. CKD was confirmed by measurements of glomerular filtration rate, blood urea nitrogen, and kidney histopathology. Mice with CKD displayed lower muscle force production and greater ischemic lesions in the tibialis anterior muscle (78.1 ± 14.5% vs. 2.5 ± 0.5% in control mice, P < 0.0001, N = 5–10/group) and decreased myofiber size (1661 ± 134 μm 2 vs. 2221 ± 100 μm 2 in control mice, P < 0.01, N = 5–10/group). This skeletal myopathy occurred despite normal capillary density (516 ± 59 vs. 466 ± 45 capillaries/20x field of view) and limb perfusion. CKD mice displayed a ~50–65% reduction in muscle mitochondrial respiratory capacity in ischemic muscle, whereas control mice had normal mitochondrial function. Hydrogen peroxide emission was modestly higher in the ischemic muscle of CKD mice, which coincided with decreased oxidant buffering. Exposure of cultured myotubes to CKD serum resulted in myotube atrophy and elevated oxidative stress, which were attenuated by mitochondrial-targeted therapies. Taken together, these findings suggest that mitochondrial impairments caused by CKD contribute to the exacerbation of ischemic pathology.

Severe skeletal muscle injuries are common and can lead to extensive fibrosis, scarring, and loss of function. Clinically, no therapeutic intervention exists that allows for a full functional restoration. As a result, both drug and cellular therapies are being widely investigated for treatment of muscle injury. Because muscle is known to respond to mechanical loading, we investigated instead whether a material system capable of massage-like compressions could promote regeneration. Magnetic actuation of biphasic ferrogel scaffolds implanted at the site of muscle injury resulted in uniform cyclic compressions that led to reduced fibrous capsule formation around the implant, as well as reduced fibrosis and inflammation in the injured muscle. In contrast, no significant effect of ferrogel actuation on muscle vascularization or perfusion was found. Strikingly, ferrogel-driven mechanical compressions led to enhanced muscle regeneration and a ∼threefold increase inmaximum contractile force of the treated muscle at 2 wk compared with no-treatment controls. Although this study focuses on the repair of severely injured skeletal muscle, magnetically stimulated bioagent-free ferrogels may find broad utility in the field of regenerative medicine.

The emerging field of regenerative cell therapy is still limited by the fewcell types that can reliably be differentiated frompluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys520 (mouse Cys533). In addition, an HIF-1α Cys520 serine mutant is resistant to 2-AAPA–induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys520 promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles.

Significance Optimal cell delivery strategies are in urgent need to enhance the specificity, efficacy, and reproducibility of cell therapy leading to minimized cell dosage and side effects. Here, we addressed this unmet need by developing injectable 3D microscale cellular niches (microniches) based on biocompatible and biodegradable gelatin microcryogels. Dramatic improvement in cell retention, survival, and therapeutic effects enabled by the primed 3D microniches was demonstrated in treatment of critical limb ischemia (CLI) in mouse models compared with the free cell-based therapy. To the best of our knowledge, this is the first convincing demonstration of injectable and primed cell delivery strategy realizing superior therapeutic efficacy with the lowest cell dosage for treating CLI in mouse model.

Angiogenesis is a complex process that depends on the delicate regulation of gene expression. Dysregulation of transcription during angiogenesis often leads to various human diseases. Emerging evidence has recently begun to show that long non-coding RNAs (lncRNAs) may mediate angiogenesis in both physiological and pathological conditions; concurrently, underlying molecular mechanisms are largely unexplored. Previously, our lab identified metastasis associates lung adenocarcinoma transcript 1 (Malat1) as an oxygen-glucose deprivation (OGD)-responsive endothelial lncRNA. Here we reported that genetic deficiency of Malat1 leads to reduced blood vessel formation and local blood flow perfusion in mouse hind limbs at one to four weeks after hindlimb ischemia. Malat1 and vascular endothelial growth factor receptor 2 (VEGFR2) levels were found to be increased in both cultured mouse primary skeletal muscle microvascular endothelial cells (SMMECs) after 16 h OGD followed by 24 h reperfusion and in mouse gastrocnemius muscle that underwent hindlimb ischemia followed by 28 days of reperfusion. Moreover, Malat1 silencing by locked nucleic acid (LNA)-GapmeRs significantly reduced tube formation, cell migration, and cell proliferation in SMMEC cultures. Mechanistically, RNA subcellular isolation and RNA-immunoprecipitation experiments demonstrate that Malat1 directly targets VEGFR2 to facilitate angiogenesis. The results suggest that Malat1 regulates cell-autonomous angiogenesis through direct regulation of VEGFR2.