Mouse Hindlimb Research Articles

SGLT2 inhibitor empagliflozin promotes revascularization in diabetic mouse hindlimb ischemia by inhibiting ferroptosis.

Gliflozins are known as SGLT2 inhibitors, which are used to treat diabetic patients by inhibiting glucose reabsorption in kidney proximal tubules. Recent studies show that gliflozins may exert other effects independent of SGLT2 pathways. In this study we investigated their effects on skeletal muscle cell viability and paracrine function, which were crucial for promoting revascularization in diabetic hindlimb ischemia (HLI). We showed that treatment with empagliflozin (0.1-40 μM) dose-dependently increased high glucose (25 mM)-impaired viability of skeletal muscle C2C12 cells. Canagliflozin, dapagliflozin, ertugliflozin, ipragliflozin and tofogliflozin exerted similar protective effects on skeletal muscle cells cultured under the hyperglycemic condition. Transcriptomic analysis revealed an enrichment of pathways related to ferroptosis in empagliflozin-treated C2C12 cells. We further demonstrated that empagliflozin and other gliflozins (10 μM) restored GPX4 expression in high glucose-treated C2C12 cells, thereby suppressing ferroptosis and promoting cell viability. Empagliflozin (10 μM) also markedly enhanced the proliferation and migration of blood vessel-forming cells by promoting paracrine function of skeletal muscle C2C12 cells. In diabetic HLI mice, injection of empagliflozin into the gastrocnemius muscle of the left hindlimb (10 mg/kg, every 3 days for 21 days) significantly enhanced revascularization and blood perfusion recovery. Collectively, these results reveal a novel effect of empagliflozin, a clinical hypoglycemic gliflozin drug, in inhibiting ferroptosis and enhancing skeletal muscle cell survival and paracrine function under hyperglycemic condition via restoring the expression of GPX4. This study highlights the potential of intramuscular injection of empagliflozin for treating diabetic HLI.

Isolated murine skeletal muscles utilize pyruvate over glucose for oxidation

IntroductionFuel sources for skeletal muscle tissue include carbohydrates and fatty acids, and utilization depends upon fiber type, workload, and substrate availability. The use of isotopically labeled substrate tracers combined with nuclear magnetic resonance (NMR) enables a deeper examination of not only utilization of substrates by a given tissue, but also their contribution to tricarboxylic acid (TCA) cycle intermediates.ObjectivesThe goal of this study was to determine the differential utilization of substrates in isolated murine skeletal muscle, and to evaluate how isopotomer anlaysis provided insight into skeletal muscle metabolism.MethodsIsolated C57BL/6 mouse hind limb muscles were incubated in oxygenated solution containing uniformly labeled 13C6 glucose, 13C3 pyruvate, or 13C2 acetate at room temperature. Isotopomer analysis of 13C labeled glutamate was performed on pooled extracts of isolated soleus and extensor digitorum longus (EDL) muscles.ResultsPyruvate and acetate were more avidly consumed than glucose with resultant increases in glutamate labeling in both muscle groups. Glucose incubation resulted in glutamate labeling, but with high anaplerotic flux in contrast to the labeling by pyruvate. Muscle fiber type distinctions were evident by differences in lactate enrichment and extent of substrate oxidation.ConclusionIsotope tracing experiments in isolated muscles reveal that pyruvate and acetate are avidly oxidized by isolated soleus and EDL muscles, whereas glucose labeling of glutamate is accompanied by high anaplerotic flux. We believe our results may set the stage for future examination of metabolic signatures of skeletal muscles from pre-clinical models of aging, type-2 diabetes and neuromuscular disease.

ENO1 expression and Erk phosphorylation in PDAC and their effects on tumor cell apoptosis in a hypoxic microenvironment.

Hypoxia is an important feature of pancreatic ductal adenocarcinoma (PDAC). Previously, we found that hypoxia promotes ENO1 expression and PDAC invasion. However, the underlying molecular mechanism was remains unclear. The relationship between ENO1 expression and clinicopathological characteristics was analyzed in 84 patients with PADC. The effects of CoCl2-induced hypoxia and ENO1 downregulation on the apoptosis, invasion, and proliferation of PDAC cells were evaluated in vitro and in vivo. Hypoxia- and ENO1-induced gene expression was analyzed by transcriptomic sequencing. The prognosis of PDAC with high ENO1 expression was poor (P < 0.05). High ENO1 expression was closely associated with histological differentiation and tumor invasion in 84 PDAC cases (P < 0.05). Hypoxia increased ENO1 expression in PDAC and promoted its migration and invasion. Apoptotic cells and the apoptosis marker caspase-3 in the CoCl2-treated ENO1-sh group were significantly elevated (P < 0.05). Transcriptomic sequencing indicated that CoCl2-induced PDAC cells initiated MAPK signaling. Under hypoxic conditions, PDAC cells upregulated ENO1 expression, thereby accelerating ERK phosphorylation and inhibiting apoptosis (P < 0.05). Consistent results were also observed in a PDAC-bearing mouse hindlimb ischemia model. Hypoxia-induced ENO1 expression promotes ERK phosphorylation and inhibits apoptosis, thus leading to PDAC survival and invasion. These results suggest that ENO1 is a potential therapeutic target for PDAC.

Loss of 4E-BPs prevents the hindlimb immobilization-induced decrease in protein synthesis in skeletal muscle.

The present study was designed to test the hypothesis that upregulating protein synthesis attenuates the loss of muscle mass in a model of disuse atrophy. The studies compared the effect of unilateral hindlimb immobilization in wild-type (WT) mice and double-knockout (DKO) mice lacking the translational regulators 4E-BP1 and 4E-BP2. Immobilization-induced downregulation of protein synthesis occurred in both groups of mice, but protein synthesis was higher in gastrocnemius muscle from the immobilized hindlimb of fasted DKO compared with WT mice. Surprisingly, although protein synthesis was partially elevated in DKO compared with WT mice, atrophy occurred to the same extent in both groups of animals. This may be partially due to impaired leucine-induced stimulation of protein synthesis in DKO compared with WT mice due to downregulated eukaryotic initiation factor eIF4E expression in muscle of DKO compared with WT mice. Expression of the E3 ubiquitin ligases MAFbx and MuRF-1 mRNAs and total protein ubiquitylation was upregulated in the immobilized compared with the nonimmobilized hindlimb of both WT and DKO mice, with little difference in the magnitude of the upregulation between genotypes. Analysis of newly synthesized proteins revealed downregulation of several glycolytic enzymes in the gastrocnemius of DKO mice compared with WT mice, as well as in the immobilized compared with the nonimmobilized hindlimb. Overall, the results suggest that the elevated rate of protein synthesis during hindlimb immobilization in fasted DKO mice is insufficient to prevent disuse-induced muscle atrophy, probably due to induction of compensatory mechanisms including downregulation of eIF4E expression.NEW & NOTEWORTHY Basal rates of protein synthesis are elevated in skeletal muscle in the immobilized leg of mice lacking the translational repressors, 4E-BP1 and 4E-BP2 (knockout mice), compared with wild-type mice. However, disuse-induced muscle atrophy occurs to the same extent in both wild-type and knockout mice suggesting that compensatory mechanisms are induced that overcome the upregulation of muscle protein synthesis. Proteomic analysis revealed that mRNAs encoding several glycolytic enzymes are differentially translated in wild-type and knockout mice.

Opposite Regulation of Homer Signal at the NMJ Postsynaptic Micro Domain between Slow- and Fast-Twitch Muscles in an Experimentally Induced Autoimmune Myasthenia Gravis (EAMG) Mouse Model

Accelerated postsynaptic remodelling and disturbance of neuromuscular transmission are common features of autoimmune neurodegenerative diseases. Homer protein isoform expression, crosslinking activity and neuromuscular subcellular localisation are studied in mouse hind limb muscles of an experimentally induced autoimmune model of Myasthenia Gravis (EAMG) and correlated to motor end plate integrity. Soleus (SOL), extensor digitorum longus (EDL) and gastrocnemius (GAS) skeletal muscles are investigated. nAChR membrane clusters were studied to monitor neuromuscular junction (NMJ) integrity. Fibre-type cross-sectional area (CSA) analysis is carried out in order to determine the extent of muscle atrophy. Our findings clearly showed that crosslinking activity of Homer long forms (Homer 1b/c and Homer2a/b) are decreased in slow-twitch and increased in fast-twitch muscle of EAMG whereas the short form of Homer that disrupts Homer crosslinking (Homer1a) is upregulated in slow-twitch muscle only. Densitometry analysis showed a 125% increase in Homer protein expression in EDL, and a 45% decrease in SOL of EAMG mice. In contrast, nAChR fluorescence pixel intensity decreased in endplates of EAMG mice, more distinct in type-I dominant SOL muscle. Morphometric CSA of EAMG vs. control (CTR) revealed a significant reduction in EDL but not in GAS and SOL. Taken together, these results indicate that postsynaptic Homer signalling is impaired in slow-twitch SOL muscle from EAMG mice and provide compelling evidence suggesting a functional coupling between Homer and nAChR, underscoring the key role of Homer in skeletal muscle neurophysiology.

Practical Mouse Model to Investigate Therapeutics for Staphylococcusaureus Contaminated Surgical Mesh Implants

IntroductionThe use of prosthetic mesh in hernia repair provides a powerful tool to increase repair longevity, decrease recurrence rates, and facilitate complex abdominal wall reconstruction. Overall infection rates with mesh are low, but for those affected there is high morbidity and economic cost. The availability of a practicable small animal model would be advantageous for the preclinical testing of prophylactics, therapeutics, and new biomaterials. To this end, we have developed a novel mouse model for implantation of methicillin-resistant Staphylococcus aureus–infected surgical mesh and provide results from antibiotic and immunotherapeutic testing. Materials and MethodsImplantation of surgical mesh between fascial planes of the mouse hind limb was used to approximate hernia repair in humans. Surgical mesh was inoculated with methicillin-resistant Staphylococcus aureus to test the efficacy of antibiotic therapy with daptomycin and/or immunotherapy to induce macrophage phagocytosis using antibody blockade of the CD47 “don't eat me” molecule. Clinical outcomes were assessed by daily ambulation scores of the animals and by enumeration of mesh-associated bacteria at predetermined end points. ResultsA single prophylactic treatment with daptomycin at the time of surgery led to improved ambulation scores and undetectable levels of bacteria in seven of eight mice by 21 days postinfection. Anti-CD47, an activator of macrophage phagocytosis, was ineffective when administered alone or in combination with daptomycin treatment. Ten days of daily antibiotic therapy begun 3 days after infection was ineffective at clearing infection. ConclusionsThis fast and simple model allows rapid in vivo testing of novel antimicrobials and immunomodulators to treat surgical implant infections.

Repair of Limb Ischemia Is Dependent on Hematopoietic Stem Cell Specific-SHP-1 Regulation of TGF-β1.

Hematopoietic stem cell (HSC) therapy has shown promise for tissue regeneration after ischemia. Therefore, there is a need to understand mechanisms underlying endogenous HSCs activation in response to ischemic stress and coordination of angiogenesis and repair. SHP-1 plays important roles in HSC quiescence and differentiation by regulation of TGF-β1 signaling. TGF-β1 promotes angiogenesis by stimulating stem cells to secrete growth factors to initiate the formation of blood vessels and later aid in their maturation. We propose that SHP-1 responds to ischemia stress in HSC and progenitor cells (HSPC) via regulation of TGF-β1. A mouse hind limb ischemia model was used. Local blood perfusion in the limbs was determined using laser doppler perfusion imaging. The number of positive blood vessels per square millimeter, as well as blood vessel diameter (μm) and area (μm2), were calculated. Hematopoietic cells were analyzed using flow cytometry. The bone marrow transplantation assay was performed to measure HSC reconstitution. After femoral artery ligation, TGF-β1 was initially decreased in the bone marrow by day 3 of ischemia, followed by an increase on day 7. This pattern was opposite to that in the peripheral blood, which is concordant with the response of HSC to ischemic stress. In contrast, SHP-1 deficiency in HSC is associated with irreversible activation of HSPCs in the bone marrow and increased circulating HSPCs in peripheral blood following limb ischemia. In addition, there was augmented auto-induction of TGF-β1 and sustained inactivation of SHP-1-Smad2 signaling, which impacted TGF-β1 expression in HSPCs in circulation. Importantly, restoration of normal T GF-β1 oscillations helped in the recovery of limb repair and function. HSPC-SHP-1-mediated regulation of TGF-β1 in both bone marrow and peripheral blood is required for a normal response to ischemic stress.

Mechanism of nerve growth factor promotes angiogenesis and skeletal muscle fiber remodeling in a mouse hindlimb ischemic model

Objective: To explore the mechanism of nerve growth factor (NGF) in the skeletal muscle fiber remodeling in ischemic limbs during therapeutic angiogenesis. Methods: Eighteen female mice with SPF grade, 6 weeks old and 25-30 g weighed were randomly allocated to sham-operated group (n=6), blank control group (n=6) and NGF gene transfection group (n=6). The left hindlimb ischemia models were established by ligating the femoral artery in blank control group and NGF gene transfection group. Seven days after the operation, mice in the three groups were separately injected with normal saline, empty plasmids, and NGF plasmids. Gastrocnemius of left hindlimbs was harvested after the blood perfusion assessment of the ischemic limb on the 21st postoperative day. The gastrocnemius muscle specimens were stained with HE, CD31 and proliferating cell nuclear antigen (PCNA) immunohistochemistry staining, the mRNA expressions of myosin heavy chain-Ⅰ(MHC-Ⅰ), MHC-Ⅱa and MHC-Ⅱb were measured by real-time PCR, and the protein level of NGF and peroxisome proliferator-activated receptors-β/δ (PPAR β/δ) were detected by Western blot. The expression of cytochrome C oxidase (COX), isocitrate dehydrogenase (IDH) and adenosine triphosphate (ATP) were examined by enzyme-linked immunosorbent assay (ELISA). Results: On the 21st day after operation, the blood perfusion of the ischemic limb in NGF gene transfection group was (195.70±9.99)PU, which was lower than that in sham-operated group (312.15±17.32)PU (P=0.001), while it was higher than that in blank control group (82.11±8.55)PU (P=0.001). The degree of muscle atrophy in the NGF gene transfection group was lower than that in the blank control group. The capillary density of NGF gene transfection group (0.34±0.05) was higher than that of sham-operated group (0.11±0.03) and blank control group (0.27±0.04) (P<0.05). The endothelial cell proliferation index in NGF gene transfection group (0.39±0.19) was significantly higher than that in sham-operated group (0.18±0.01) and blank control group (0.25±0.14) (P<0.05). The expression of NGF, PPAR β/δ, COX, IDH, ATP, and MHC-Ⅰ mRNA in NGF gene transfection group were significantly higher than those in sham-operated group and blank control group (P<0.05). Conclusions: NGF gene transfection can promote angiogenesis in the ischemic limbs of mice, increase the blood perfusion, and thus induce the remodeling of skeletal muscle fibers to type Ⅰ. This process may be related to NGF-induced PPAR β/δ expression and promote the cellular aerobic metabolism in skeletal muscle.

Huanglian Jiedu plaster ameliorated X-ray-induced radiation dermatitis injury by inhibiting HMGB1-mediated macrophage-inflammatory interaction

Ethnopharmacological relevanceHuanglian Jiedu plaster (HJP) is a kind of Chinese patent medicine that contains four medicinal plants. It has been clinically proven to be beneficial for the treatment of tumor-associated radiation dermatitis. However, the underlying mechanism of HJP on radiation dermatitis remains unclear. Aim of the studyThis study aims to investigate the therapeutic effect of HJP on X-ray-induced radiation dermatitis, and how HJP improves the inflammatory response and skin damage of radiation dermatitis. Materials and methodsIn this study, We selected a case of esophageal cancer as a clinical demonstration of the efficacy of radiation dermatitis. The patient received a total radiation dose of 7000cGY, and treatment by HJP for 14 days.RD mouse models were established through continuous irradiation with X-ray (800cGY) on the right hind limb of mice for 5 days, and the treatment group mice was applied HJP to the irradiated skin for 15 days from modeling. An inflammatory cellular model was induced through irradiation with X-ray (100cGY) in JB6 cells and a co-culture system of JB6 cell and macrophage was established to examine the effect and mechanism of HJP on the inflammatory interaction of these two cells. The activation of HMGB1-TLR4-NF-κB signaling pathway, and the levels of epidermal injury related factors and inflammatory cytokins were subsequently detected. ResultsThe results showed that HJP can significantly alleviate X-ray-induced skin injury, inhibiting skin inflammation and reducing the expression of inflammatory cytokins (IL-1β, IL-6, TNF-α) and epidermal damage related factors (Integrin β1, CXCL9 and Cytokeratin17), as well as significantly down-regulated the protein level of HMGB1 (a key DAMPs factor) in vivo and in vitro. Cell co-culture experiments demonstrated that HMGB1 released from X-ray-induced JB6 cells can promote inflammatory response of macrophage, which then feedback aggravate epithelial cell damage, notably, HJP can significantly improve radiation skin lesion by inhibiting HMGB1-mediated inflammatory interaction between epithelial cells and macrophages. ConclusionIn summary, these findings indicated the role of HJP in the treatment of RD by inhibiting the inflammatory interaction between macrophage and JB6 cells mediated by HMGB1, which may provide a reliable therapeutic method for RD. Furthermore, HMGB1 may be an effective target for HJP to inhibit inflammation and ameliorate skin damage in RD.

Nanoceria-GO-intercalated multicellular spheroids revascularize and salvage critical ischemic limbs through anti-apoptotic and pro-angiogenic functions

Critical limb ischemia (CLI) is a serious form of peripheral arterial disease that involves severe blockage of blood flow in lower extremities, often leading to foot necrosis and limb loss. Lack of blood flow and high pro-inflammation with overproduced reactive oxygen species (ROS) in CLI aggravate the degenerative events. Among other therapies, cell delivery is considered potential for restoring regenerative capacity, and preservation of cell survival under high oxidative stress has been challenging and prerequisite to harness cellular functions. Here, we introduce a multicellular delivery system that is intercalated with nanoceria-decorated graphene oxide (CeGO), which is considered to have high ROS scavenging ability while providing cell-matrix interaction signals. The CeGO nano-microsheets (8-nm-nanoceria/0.9-μm-GO) incorporated in HUVEC/MSC (7/3) could form cell-material hybrid spheroids mediated by cellular contraction. Under in vitro oxidative-stress-challenge with H2O2, the CeGO-intercalation enhanced the survival and anti-apoptotic capacity of cellular spheroids. Pro-angiogenic events of cellular spheroids, including cell sprouting and expression of angiogenic markers (HIF1α, VEGF, FGF2, eNOS) were significantly enhanced by the CeGO-intercalation. Proteomics analysis also confirmed substantial up-regulation of a series of angiogenesis-related secretome molecules. Such pro-angiogenic events with CeGO-intercalation were proven to be mediated by the APE/Ref-1 signaling pathway. When delivered to ischemic hindlimb in mice, the CeGO-cell spheroids could inhibit the accumulation of in vivo ROS rapidly, preserving high cell survival rate (cells were more proliferative and less apoptotic vs. those in cell-only spheroids), and up-regulated angiogenic molecular expressions. Monitoring over 28 days revealed significantly enhanced blood reperfusion and tissue recovery, and an ultimate limb salvage with the CeGO-cell delivery (∼60% salvaged vs. ∼29% in cell-only delivery vs. 0% in ischemia control). Together, the CeGO intercalated in HUVEC/MSC delivery is considered a potential nano-microplatform for CLI treatment, by scavenging excessive ROS and enhancing transplanted cell survival, while stimulating angiogenic events, which collectively help revascularization and tissue recovery, salvaging critical ischemic limbs.

C1q/TNF-related protein-2 improved angiogenesis to protect myocardial function during ischaemia‒reperfusion.

Collateral growth plays an important role in the recovery of acute myocardial infarction. C1q/TNF-related protein-2 (CTRP2), a CTRP family member, showed some protective effects on cell survival. In this study, the relationship between CTRP2 and collateral growth was examined. C57BL/6 mice were subjected to myocardial ischaemia/reperfusion (I/R), and the expression of CTRP2 and the effect of CTRP2 on infarction size, cardiac function and angiogenesis were examined. The ischaemic hindlimb model was also used to examine the effect of CTRP2. In vitro, CTRP2-mediated regulation of angiogenesis, AKT activation and VEGFR2 expression in endothelial cells was examined. The CTRP2 level associated with good collateral growth was observed in a cohort. I/R reduced CTRP2 expression, and intraperitoneal injection of recombinant CTRP2 protein improved infarction size, cardiac function and angiogenesis. Overexpression of CTRP2 promoted blood refusion and collateral growth in ischaemic hindlimb mice. In vitro, CTRP2 enhanced tube formation and migration in a dose-dependent manner, while CTRP2 increased AKT phosphorylation and VEGFR2 expression. In an observational clinical cohort, CTRP2 levels were significantly increased in patients with good collateral growth, and CTRP2 was negatively associated with poor collateral growth in patients. CTRP2 improved cardiac function by promoting collateral growth by promoting AKT-VEGFR2.

Single and combined impacts of irradiation and surgery on lymphatic vasculature and fibrosis associated to secondary lymphedema.

Lymphedema (LD) refers to a condition of lymphatic dysfunction associated with excessive fluid accumulation, fibroadipose tissue deposition and swelling. In industrialized countries, LD development mainly results from a local disruption of the lymphatic network by an infection or cancer-related surgery (secondary LD). In the absence of efficient therapy, animal models are needed to decipher the cellular and molecular mechanisms underlying LD and test putative drugs. In this study, we optimized and characterized a murine model of LD that combines an irradiation of the mice hind limb and a radical surgery (lymph node resection associated to lymphatic vessel ligation). We investigated the respective roles of irradiation and surgery in LD formation by comparing their impacts, alone or in combination (with different intervention sequences), on eight different features of the pathology: swelling (paw thickness), indocyanine green (ICG) clearance, lymphatic vasculature remodeling, epidermal and dermal thickening, adipocyte accumulation, inflammatory cell infiltration and collagen deposition. This study supports the importance of radiation prior to surgery to experimentally induce a rapid, severe and sustained tissue remodeling harboring the different hallmarks of LD. We provide the first experimental evidence for an excessive deposition of periostin (POSTN) and tenascin-C (TNC) in LD. Through a computerized method of digital image quantification, we established the spatial map of lymphatic expansion, as well as collagen, POSTN and TNC deposition in papillary and reticular dermis of lymphedematous skins. This mouse model is available to study the patho-physiology of LD and test potential therapeutic targets.

Assessment of PABPN1 nuclear inclusions on a large cohort of patients and in a human xenograft model of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a rare muscle disease characterized by an onset of weakness in the pharyngeal and eyelid muscles. The disease is caused by the extension of a polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) protein leading to the formation of intranuclear inclusions or aggregates in the muscle of OPMD patients. Despite numerous studies stressing the deleterious role of nuclear inclusions in cellular and animal OPMD models, their exact contribution to human disease is still unclear. In this study, we used a large and unique collection of human muscle biopsy samples to perform an in-depth analysis of PABPN1 aggregates in relation to age, genotype and muscle status with the final aim to improve our understanding of OPMD physiopathology. Here we demonstrate that age and genotype influence PABPN1 aggregates: the percentage of myonuclei containing PABPN1 aggregates increases with age and the chaperone HSP70 co-localize more frequently with PABPN1 aggregates with a larger polyalanine tract. In addition to the previously described PRMT1 and HSP70 co-factors, we identified new components of PABPN1 aggregates including GRP78/BiP, RPL24 and p62. We also observed that myonuclei containing aggregates are larger than myonuclei without. When comparing two muscles from the same patient, a similar amount of aggregates is observed in different muscles, except for the pharyngeal muscle where fewer aggregates are observed. This could be due to the peculiar nature of this muscle which has a low level of PAPBN1 and contains regenerating fibers. To confirm the fate of PABPN1 aggregates in a regenerating muscle, we generated a xenograft model by transplanting human OPMD muscle biopsy samples into the hindlimb of an immunodeficient mouse. Xenografts from subjects with OPMD displayed regeneration of human myofibers and PABPN1 aggregates were rapidly present—although to a lower extent-after muscle fiber regeneration. Our data obtained on human OPMD samples add support to the dual non-exclusive models in OPMD combining toxic PABPN1 intranuclear inclusions together with PABPN1 loss of function which altogether result in this late-onset and muscle selective disease.

Pencil beam scanning proton FLASH maintains tumor control while normal tissue damage is reduced in a mouse model.

Preclinical studies indicate a normal tissue sparing effect when ultra-high dose rate (FLASH) radiation is used, while tumor response is maintained. This differential response has promising perspectives for improved clinical outcome. This study investigates tumor control and normal tissue toxicity of pencil beam scanning (PBS) proton FLASH in a mouse model. Tumor bearing hind limbs of non-anaesthetized CDF1 mice were irradiated in a single fraction with a PBS proton beam using either conventional (CONV) dose rate (0.33-0.63Gy/s field dose rate, 244MeV) or FLASH (71-89Gy/s field dose rate, 250MeV). 162 mice with a C3H mouse mammary carcinoma subcutaneously implanted in the foot were irradiated with physical doses of 40-60Gy (8-14 mice per dose point). The endpoints were tumor control (TC) assessed as no recurrent tumor at 90days after treatment, the level of acute moist desquamation (MD) to the skin of the foot within 25days post irradiation, and radiation induced fibrosis (RIF) within 24weeks post irradiation. TCD50 (dose for 50% tumor control) was similar for CONV and FLASH with values (and 95% confidence intervals) of 49.1 (47.0-51.4) Gy for CONV and 51.3 (48.6-54.2) Gy for FLASH. RIF analysis was restricted to mice with tumor control. Both endpoints showed distinct normal tissue sparing effect of proton FLASH with MDD50 (dose for 50% of mice displaying moist desquamation) of <40.1Gy for CONV and 52.3 (50.0-54.6) Gy for FLASH, (dose modifying factor at least 1.3) and FD50 (dose for 50% of mice displaying fibrosis) of 48.6 (43.2-50.8) Gy for CONV and 55.6 (52.5-60.1) Gy for FLASH (dose modifying factor of 1.14). FLASH had the same tumor control as CONV, but reduced normal tissue damage assessed as acute skin damage and radiation induced fibrosis.

LncRNA PVT1 is a novel mediator promoting the angiogenesis response associated with collateral artery formation.

Angiogenesis plays a key role in coronary collateral circulation (CCC), the compensatory formation of new blood vessels during chronic total coronary occlusion. This study aimed to determine whether plasmacytoma variant translocation 1 (PVT1), a long non-coding (lnc) RNA involved in tumor angiogenesis, plays a role in regulating angiogenesis during chronic coronary ischemia. Patients with coronary artery disease, and ≥90% stenosis, were examined and divided into "Good" and "Poor" CCC groups based on Rentrop Cohen classification. RNA samples were obtained from all patients, as well as from oxygen and glucose-deprived (OGD) HUVECs. PVT1, miR-15b-5p and AKT3 levels were measured with RT-qPCR or Western blot, while HUVEC migration and angiogenesis were detected by, respectively, wound-healing and tube formation assays. Luciferase reporter assay confirmed direct PVT1-miR-15b-5p binding. Increased PVT1 was found in "Good CCC" patient plasma, along with being highly expressed among OGD HUVECs; PVT1 knockdown reduced HUVEC migration, tube formation, and pro-angiogenic factor expression. Conversely, OGD HUVECs had downregulated miR-15b-5p, and miR-15b-5p overexpression significantly depressed their angiogenic capabilities. These PVT1 knockdown- or miR-15b-5p overexpression-associated reductions in angiogenic effects were reversed by AKT3 overexpression. In vivo, neovascularization and functioning in both ischemic mice hind-limbs and infarcted myocardium injected with ADV-sh-PVT1 were reduced, which were ameliorated by concurrent antagomiR-15b-5p injections. Circulating PVT1 may serve as a useful biomarker to distinguish between good versus poor CCC, as it is involved in orchestrating angiogenesis via the miR-15b-5p-AKT3 axis; it thus has potential as a target for treating ischemic disease.

Cannabinoid receptor-2 attenuates neuroinflammation by promoting autophagy-mediated degradation of the NLRP3 inflammasome post spinal cord injury.

BackgroundNeuroinflammation following spinal cord injury (SCI) results in prolonged neurological damage and locomotor dysfunction. Polarization of microglia is vital to regulation of neuroinflammation, although the underlying mechanisms have not yet been elucidated. Endocannabinoid receptor subtype 2 (CB2R) is reported to ameliorate neurodegeneration via immunomodulation activities. However, the underlying machinery in the context of SCI remains unclear.MethodsA lipopolysaccharide-induced microglia inflammation model and a mouse model of SCI were employed to investigate the regulatory role of CB2R in the polarization of microglia in response to excess neuroinflammation. Markers of inflammation and autophagy were measured by Western blot analysis, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. Histological staining with hematoxylin and eosin, Nissl, and Luxol® fast blue was conducted using commercial kits. The locomotor function of the hindlimbs of the experimental mice was evaluated with the Basso Mouse Scale, Louisville Swim Scale, and footprint assay.ResultsThe results showed that CB2R promoted M2 differentiation, increased interleukin (IL)-10 expression, and inhibited M1 differentiation with decreased expression of IL-1β and IL-6. CB2R activation also increased ubiquitination of the NLRP3 inflammasome and interacted with the autophagy-related proteins p62 and microtubule-associated proteins 1B light chain 3. Treatment with the CB2R activator JWH-133 reduced loss of myelin, apoptosis of neurons, and glial scarring, leading to improved functional recovery of the hindlimbs, while the CB2R antagonist AM630 produced opposite results.ConclusionTaken together, these results suggested that CB2R activation attenuated neuroinflammation targeting microglial polarization by promoting NLRP3 clearance, thereby facilitating functional recovery post-SCI.

Zinc chelator treatment in crush syndrome model mice attenuates ischemia–reperfusion-induced muscle injury due to suppressing of neutrophil infiltration

In crush syndrome, massive muscle breakdown resulting from ischemia–reperfusion muscle injury can be a life-threatening condition that requires urgent treatment. Blood reperfusion into the ischemic muscle triggers an immediate inflammatory response, and neutrophils are the first to infiltrate and exacerbate the muscle damage. Since free zinc ion play a critical role in the immune system and the function of neutrophils is impaired by zinc depletion, we hypothesized that the administration of a zinc chelator would be effective for suppressing the inflammatory reaction at the site of ischemia–reperfusion injury and for improving of the pathology of crush syndrome. A crush syndrome model was created by using a rubber tourniquet to compress the bilateral hind limbs of mice at 8 weeks. A zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) was administered immediately after reperfusion in order to assess the anti-inflammatory effect of the chelator for neutrophils. Histopathological evaluation showed significantly less muscle breakdown and fewer neutrophil infiltration in TPEN administration group compared with control group. In addition, the expression levels of inflammatory cytokine and chemokine such as IL-6, TNFα, CXCL1, CXCL2, CXCR2, CCL2 in ischemia–reperfusion injured muscle were significantly suppressed with TPEN treatment. Less dilatation of renal tubules in histological evaluation in renal tissue and significantly better survival rate were demonstrated in TPEN treatment for ischemia–reperfusion injury in crush syndrome. The findings of our study suggest that zinc chelators contributed to the resolution of exacerbation of the inflammatory response and attenuation of muscle breakdown in the acute phase after crush syndrome. In addition, our strategy of attenuation of the acute inflammatory reaction by zinc chelators may provide a promising therapeutic strategy not only for crush syndrome, but also for other diseases driven by inflammatory reactions.

BAG3 Attenuates Ischemia-Induced Skeletal Muscle Necroptosis in Diabetic Experimental Peripheral Artery Disease.

Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, resulting in ischemic limb injuries. Individuals with diabetes and PAD typically have more severe ischemic limb injuries and limb amputations, but the mechanisms involved are poorly understood. Previously, we identified BAG3 as a gene within a mouse genetic locus termed limb salvage QTL1 on mouse chromosome 7 that determined the extent of limb necrosis following ischemic injury in C57Bl/6 mice. Whether BAG3 deficiency plays a role in the severe ischemic injury observed in diabetic PAD is not known. In vitro, we found simulated ischemia enhanced BAG3 expression in primary human skeletal muscle cells, whereas BAG3 knockdown increased necroptosis markers and decreased cell viability. In vivo, ischemic skeletal muscles from hind limbs of high-fat diet (HFD)-fed mice showed poor BAG3 expression compared to normal chow diet (NCD)-fed mice, and this was associated with increased limb amputations. BAG3 overexpression in ischemic skeletal muscles from hind limbs of HFD mice rescued limb amputation and improved autophagy, necroptosis, skeletal muscle function and regeneration. Therefore, BAG3 deficiency in ischemic skeletal muscles contributes to the severity of ischemic limb injury in diabetic PAD, likely through autophagy and necroptosis pathways.

Increasing angiogenic efficacy of conditioned medium using light stimulation of human adipose-derived stem cells

Conditioned medium (CM) contains various therapeutic molecules produced by cells. However, the low concentration of therapeutic molecules in CM is a major challenge for successful tissue regeneration. Here, we aim to develop a CM enriched in angiogenic paracrine factors for the treatment of ischemic diseases. Combining spheroidal culture and light irradiation significantly upregulates the angiogenic factor expression in human adipose-derived stem cells (hADSCs). Spheroids of light-irradiated hADSCs (SR group) show significantly enhanced expression of angiogenic paracrine factors compared with spheroids without light stimulation. Enhanced viability, migration, and angiogenesis are observed in cells treated with CM derived from the SR group. Furthermore, we performed in vivo experiments using a mouse hindlimb ischemia model; the results demonstrate that CM derived from densely cultured spheroids of light-irradiated hADSCs induced increased angiogenesis in vivo. In conclusion, our proposed approach of using light to stimulate stem cells may overcome the major drawbacks of CM-based therapies.

MicroRNA-375 repression of Kruppel-like factor 5 improves angiogenesis in diabetic critical limb ischemia.

Peripheral artery disease (PAD) is an occlusive disease of limb arteries. Critical limb ischemia (CLI) is an advanced form of PAD that is prognostically worse in subjects with diabetes and can result in limb loss, gangrene, and death, although the underlying signaling mechanisms that contribute to its development remain poorly understood. By comparing plasma samples from diabetic humans with PAD and mouse models of PAD, we identified miR-375 to be significantly downregulated in humans and mice during progression to CLI. Overexpression of miR-375 was pro-angiogenic in endothelial cells in vitro and induced endothelial migration, proliferation, sprouting, and vascular network formation, whereas miR-375 inhibition conferred anti-angiogenic effects. Intramuscular delivery of miR-375 improved blood flow recovery to diabetic mouse hindlimbs following femoral artery ligation (FAL) and improved neovessel growth and arteriogenesis in muscle tissues. Using RNA-sequencing and prediction algorithms, Kruppel-like factor 5 (KLF5) was identified as a direct target of miR-375 and siRNA knockdown of KLF5 phenocopied the effects of miR-375 overexpression in vitro and in vivo through regulatory changes in NF-kB signaling. Together, a miR-375-KLF5-NF-kB signaling axis figures prominently as a potential therapeutic pathway in the development CLI in diabetes.